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1. ade d in Fal a 1 Fa m TU VT Fida l 7 h db Vikram Shankar Adam Shutes Bill Griffin Brian Healey Lead Discovery Technologies EMD Serono Research Institute Rockland MA 02370 vikram shankar emdserono com low volume liquid handling Evaluation of the Formulatrix Tempest at EMD Serono A flexible low volume handler with two axis gradient capability Liquid handling instrumentation which provides precise and flexible dispensing over arange of volumes and different components is core to a fully functional assay development hit discovery and characterisation lab At EMD Serono we use liquid dispensing 50 ul to 200 nl in a limited number of tasks such as regular assay component dispensing around 15 70 ul as well as low volume dispensing 200 nl Other aspects of assay development amp biochemical characterisation of inhibitors are performed in a manual and labour intensive manner Introducing the Formulatrix Tempest in our workflow has allowed us to improve the current technique not only in terms of but also accuracy and precision Unique to the Te zn stis 15 microfluidic technology that relies on indem dent channel all the 96 nozzles thereby allowing one to dispense any volume f input into any well We evaluated thi
2. E x l 1 i E 4 T bir ud or Fd ee zh da P T ED m ER d LF Lik m E 4 en i 1 7 h lt tat m u Pima TBE Kar 22 z i 1 LL S BEN Through Compact Automation EJ LESS Vo E um odel system X A Universal Mass Specttometric A ImmunoasSay k i Ji 5 p me m 1 EJ A E fe E we T 4 Ea SCIENTIFIC The world leader in serving science Offices amp Service Centres Welcome Australia Head Office Street Address 5 Caribbean Dr Scoresby Victoria 3179 Postal Address PO Box 9092 Scoresby Vic 3179 Telephone Number 1300 735 292 AU Service Centre Locations Melbourne Scoresby Sydney North Ryde Brisbane Richlands South Australia Thebarton Perth Malaga New Zealand Head Office Street Address 244 Bush Road Albany North Shore City 0632 Postal Address Private Bag 102922 North Shore North Shore City 0745 Telephone Number 0800 933 966 NZ Service Centre Locations Auckland Albany Christchurch Wigram Palmerston North Wellington Lower Hutt Editor Mika Mitropoulos mika mitro
3. luctivity Compact automation References 1 Heavy atoms or groups of atoms shift the IR wavenumber value lower according to the relationship where v is the IR wavenumber cm and is the reduced mass As the mass increases the IR peak shifts to lower wavenumbers vav1 u Glossary CaF calcium fluoride DLaTGS deuterated L alanine doped triglycene sulphate InGaAs Indium gallium arsenide KBr potassium bromide Many modern analytical laboratories face increasing workloads from a broad range of sample types with a simultaneous arive for faster results and more complex sample characterisation needs Many routine QC QA laboratories can perform material analyses with single range basic Fourier transform infrared FT IR instrument configurations However modern analytical laboratories face increasing workloads from a broad range of sample types with a simultaneous drive for faster results and more complex sample characterisation needs Flexibility to analyse multiple sample types becomes mandatory when rapidly responding to these different application requests Such diversity requires laboratory instruments to be reconfigured for specific measurements multiple times per day taking time away from other critical activities This also implies that laboratory personnel possess the necessary skills experience to make decisions how best to configure the instru
4. T arae n ee ee ke R Accelerate Advance Achieve he tools amp technologies of p M The Thermo Scientific myECL Imager revolutionises image acquisition of protein and nucleic acid blots and gels detected via chemiluminescent colorimetric or UV light activated fluorescent substrates or stains The compact benchtop instrument uses UV and visible light transillumination specialised filters and advanced CCD camera technology to capture i images with high sensitivity and dynamic range The large 10 4 hon touchscreen and on board computer Bes 4 elegant the instrument is a five computer Scientific mylmageAnalysis Software ac powerfulanalysistooc 1 WA Lynx Centrifuge This ntrifuge oif S is exceptional rforma to mee thee ap plication of academic and ites With its Ju DEN high throughput sample pr sing built in safety and efficiency and proven r yt TSU Ultra Low Temperature Freezer Thermo Scientific TSU Series freezers deliver ultimate protection and optimum capacity for your most critical samples The TSU Series achieve outstanding thermal performance safety and security through state of the art engineering Packed full of features the TSU Series has new Cabinet Vacuum Insulation Panel Technology which increases Sorv
5. 10 11 12 Add 15 mL buffer mix by inversion 5 6 times and let sit at room temperature for 5 min Solution should become more clear and viscous Add 15 mL buffer C mix by inversion 5 6 times A heavy precipitate will form swirl to break up precipitate Centrifuge at 20 000 x g for 20 min at 4 C Pour supernatant through cheese cloth and collect in a clean bottle Note An additional spin for 10 min may pellet any excessive debris not cleared in the first spin Add an equal amount of isopropanol to the bottle and swirl gently to mix Perform centrifugation with the following parameters 18 000 x g for 30 min at 4 C Note Use alab pen to mark the outer edge of the bottle before centrifugation to help locate the somewhat clear and translucent pellet also applies to subsequent steps Carefully remove the supernatant Add gt 50 mL of 7096 ethanol to the 250 mL bottle and agitate to resuspend and wash the DNA Perform centrifugation with the following parameters 18 000 x g for 10 min at 4 Remove all the supernatant and dry the pellet in a vacuum dessicator for 30 60 min Over drying the pellet can prevent effective DNA resuspension in subsequent steps Dissolve the pellet in a total of 2 mL TE containing RNAase at a concentration of 100 ug mL If dissolved in higher volumes the DNA may be too dilute to effectively precipitate in subsequent steps Transfer to a 15 mL or similar volume tube and incubate at
6. 10 11 12 2 34 8 T 8 3 Time 8 using Thermo Scientific luminometers for performing Pierce Luciferase Reporter Assays include the following e Excellent luminometric performance in glow flash ype assays Onboard dispensers for reagent addition follow up of fast kinetics of flash assays precise timing between dispensing and measurement in flash assays convenience and accuracy for flash and glow assays Optimised filters for measurement of dual assays e Flexible and easy protocol setup and data processing with PC software 120 1 10 0084 0 70 0 60 0 50 ee 12 34 58 T 8 301 12 Time Kinetics of Firefly flash assay gt Figure 3 Luciferase reaction kinetics of flash type assays measured both in a normal 384 well plate and a shallow well 384 plate Renilla and Firefly luciferases were measured from a cell lysate Gaussia and Cypridina were secreted to the growth medium and measured directly from the medium without a cell lysis TR 2 39 4 www thermofisher co nz bio innovation www thermofisher com au bi novation Irmgard Suominen Senior Application Scientist Thermo Fisher Scientific Vantaa Finland Sami Koivisto Technology Manager Liquid Transfer Thermo Fisher Scientific Vantaa Finland Suvi Bergh ll Product Manager Pipetting Systems Thermo Fisher Scientific
7. LIT T Figure 4 0 ye Bio degradabia Polymer Far Pt Lh Is Figure 2 Mid IR and far IR spectra of Acetylferrocene The far IR optics permit collection to 1700 cm which may be sufficient fingerprint and far IR for many applications Figure 3 The Thermo Scientific Nicolet i550 FT IR spectrometer configured for FT Raman near IR and mid far IR ATR with the automated beamsplitter exchanger Figure 4 Touch Points on the Nicolet i550 spectrometer employ one button switching between modules and the 1550 ABX automates optics set up Touch Point A NIR module Touch Point B Raman module Touch Point C Built in diamond ATR Component D ABX Automated Beamsplitter Exchanger Figure 5 Multi technique data for a recyclable plastic component using the spectrometer pictured in Figure 3 Inset shows NIR independently for clarity 111 Immunoaffinity capture and enrichment of protein and peptide targets is a widely utilised method for sample cleanup prior to mass spectrometric analysis Termed the Mass Spectrometric Inmunoassay MSIA this hybridised combination of micro scale purification with highly sensitive detection has repeatedly demonstrated the ability to F detect much lower abundance proteins from g given proteome as compared to other fractionation methods Eric E Niederkofler Senior Scientist David A Phillips Technician
8. Results 1 Tip attachment Attachment force 3 8 kg was sufficient to attach tips to all pipettes tested However tips from Manufacturer B and generic tips fell off during pipetting when 8 rows of the microplate had been dispensed Figure 2 shows ClipTip tips and Generic pipette tips after filling two 96 well microplates The ClipTip pipette tips did not fall off and secured an equal liquid level in all 12tips Amm Figure 2 ClipTip tips and Generic tips b after dispensing into two 96 well microplates The tip attachment force used was 3 8 kg Generic tips had one tip fall off and obtained uneven liquid levels in the remaining tips In order to keep tips attached the force needed to be increased from 3 8 kg to 4 8 kg The tips of manufacturer A stayed attached but the variation between dispensed volumes of different channels was higher than with F1 ClipTip Fig 3 This variation could not be observed visually 2 Precision Precision value of manufacturer A was 6896 higher than with F1 ClipTip system Figure 4 after dispensing into two 96 well microplates The figure shows that with F1 ClipTip the variation between channels was lower than with a pipette with a friction based tip fitting mechanism With friction based mechanisms the precision varied with the tip attachment force used Summary An optimal pipette and tip system increases confidence i
9. Ab dA 0 0 8 9 hy rn Re gt get attracted to great results m pum au memp ee ee ooo p F E 8 A b Ea manual protocol automated protocol M 1 2 3 4 5 1 2 3 4 5 M MagJET Kits www thermofisher co nz bio innovation www thermofisher com au bio innovation gt With pharmaceutical companies investing heavily in extensive R amp D to produce effective vaccines for existing and emerging health risks there Is a growing recognition that protecting the potency and viability of a vaccine during storage Is essential Effect One of the most rapidly expanding pharmaceutical markets today the vaccine therapy category protects public health globally and saves an estimated three million lives each year However incorrect refrigeration and storage of vaccines can lead to costly mistakes In clinical practice refrigeration problems have been cited as a major cause of the 20 million wasted each year from ruined vaccines in the U S Federal Vaccines for Children Program 2 In addition a recent UK study revealed that 4096 of vaccines were kept at the incorrect temperature which could potentially damage a vaccine s potency and effectiveness Because itis not apparent if a vaccine has become sub standard through exposure to temperature fluctuations
10. Dobrin Nedelkov Manager of Research and Development and Urban A Kiernan Senior Scientist Thermo Fisher Scientific Tempe Arizona Bryan Krastins Senior Applications Scientist Scott Peterman Senior Applications Scientist Alejandra Even though this approach to sample purification is now the staple in proteomics methods more robust consistent and Canter Thermo Fisher Scientific versatile methods for performing this sample purification and enrichment step are a growing necessity To further improve upon this front end sample processing new technology Thermo Scientific V MSIA was tested In the form of a functional pipettor tip their performance was evaluated see Application Notes MSIA1002 and MSIA1003 and bench marked against other technologies However to perform these tests model system translatable to various ligand surfaces including Protein A Protein G and Protein A G was needed Presented are the results of a developed model system for the Protein A G MSIA Tips based on Insulin Like Growth Factor 1 IGF1 a clinically relevant endocrine and oncology protein Garces Senior Applications Scientist and Mary F Lopez BRIMS Center Director BRIMS Cambridge Massachusetts Figure 1 Protein A G MSIA Tip Workflow Protein A G MSIA Tips are used by repetitive pipetting of solutions allowing for efficient interaction between capture and analyte reagents withi
11. SINA DU Nucleic acid isolation being one of the most fundamental steps in molecular biology applications is a prerequisite for successful experimental workflow MagJET nucleic acid purification kits magnetic bead based technology allowing for highly efficient nucleic acid isolation from a variety of samples at any throughput MagJET kits provide high purity DNA and RNA ready to use in routine and demanding downstream applications Dual magnetic core for fast magnetic response 1Jm 1090 Magnetite content 60 Automated and manual workflows MagJET nucleic acid isolation technology is suitable both for high throughput automated and manual sample processing The automation protocols are optimised for Thermo Scientific KingFisher instruments where magnetic beads transferred from well to well during the purification procedure In the manual protocol a magnetic field applied by an external source is used to capture the beads against the wall of the tube The supernatant is removed by aspiration Robust Performance Manual and automated workflows were used to purify RNA from N tabacum leaf samples each 50 mg with the MagJET Plant RNA The Thermo Scientific KingFisher protocol was used for the automated workflow Isolated RNA was analysed by agarose gel electrophoresis 190 M Thermo Scientific RiboRuler High Range RNA Ladder Cat 5 1823
12. incorrect storage temperatures can lead to a very real danger that patients are given a sub standard vaccine leaving them unprotected against dangerous diseases Storing vaccines in controlled and constant temperature conditions is therefore the key to protecting a population at risk Correct vaccine refrigeration also reduces expensive wastage of high value and fragile biological samples eliminating the need for costly revaccination programmes that may have to be initiated if the integrity of a vaccine becomes compromised gt Choosing correct storage There are significant differences in levels of performance and reliability across various categories of cold storage equipment Critical cold storage applications demand a safe stable environment the temperature fluctuations found in household or commercial cabinets must be eliminated Choosing the appropriate equipment with proven performance that meets all government standards is now an important business decision for research hospital pharmacy and clinical laboratories Today s specialised laboratory refrigerators and freezers are well equipped to provide efficient high value biological sample storage that maintains vaccine integrity Improvements in design and technology ensure that advanced refrigeration can keep vaccines at the constant conditions that clinics and laboratories require Maintaining constant temperature The majority of commonly administered vaccines
13. mih Chal ten Bertie bs Irem manor 4 Figure 4 Precision values with 12 channel pipettes after dispensing into two 96 well microplates The set volume was 100 ul and the tip attachment forces used were as 3 8 kg NM NH ibe severe contamination risk particularly in clinical applications With Manufacturer and more effort was needed to attach the tips firmly and prevent tips getting loose or dropping off The complete seal between the sealing ring and the ClipTip tips guaranteed optimal pipetting performance as demonstrated by the excellent precision The unique interlocking tip attachment mechanism ensured that the tips stayed firmly attached through the entire application These innovative features make F1 ClipTip Pipetting System an excellent choice for microplate applications by saving valuable samples reagents as well as time and resources while increasing confidence in pipetting results In addition the ergonomic benefits of this System reduce the risk of Repetitive Strain Injuries while pipetting 4 Issue 7 d P tor Thermo Scientific Ultra Low Temperature Freezers Freezer farms with ultra low freezers are very common in academic institutions sample storage areas biopharma sites and many other locations where ultra low temperatures are requ
14. peptides Figure 4 We also characterised an anti IMT antibody developed against the reporter region of the TMT reagent for immuno enrichment of iodo TM T labelled peptides Figure 5 and Western blot detection of iodo T MT labelled proteins Figure We used the iodoT MT reagent as a probe for labeling o nitrosylated cysteines in a modified S nitro switch assay Figure 6 lodo T MT reagents successfully labelled S nitrosylated cysteines after selective reduction using ascorbate however labelling efficiency was less than MT reagents This result is consistent with different efficiencies of the sulfhyrdyl reactive groups dithiopryridine vs iodoacetyl of each reagent In addition we discovered that addition of 1mM copper sulfate to the switch reaction buffer inhibited iodo T MT reagent labeling but not reagent labeling Figure 5 Donga o of Th Hiri Figure 6 iodoTIMT Addition of copper sulfate is thought to facilitate S NO bond reduction during the labeling reaction however in the presence of ascorbate Cu is readily reduced to which can generate free radicals The free radicals generated resulted in protein degradation Figure 6B lanes 5 8 12 and 15 and possible loss of iodine from the iodoacetyl reactive group Overall using the combination of iodo IMT labeling with anti IMT enrichment has several advantages over p
15. GKSB 314 were purchased from Prozyme Hayward Prior to analysis dissolve samples in 10096 buffer 100 mM ammonium formate 4 4 and dilute further with acetonitrile to make 3096 buffer and 70 acetonitrile Liquid Chromatography All the glycans were separated using a Thermo Scientific M Dionex UltiMate 3000 BioRS system and either a Thermo Scientific M Dionex M UltiMate M FL D 3400RS Separation Fluorescence Detector or MS detector Mass Spectrometry MS analysis was performed with a Thermo Scientific M Q Exactive Benchtop LC MS MS in negative ion mode at the following settings MS scan range 380 2000 FI MS was acquired at 70 000 resolution at m z 200 with AGC target of 1e5 DDA MS2 acquired at 17 500 resolution at m z 200 with AGC target of 2e Data Analysis SimGlycan software PREMIER Biosoft was used for glycan identification and structural elucidation data analysis SimGlycan software accepts raw data files from Thermo Scientific mass spectrometers and elucidates the associated glycan structure by database searching and scoring techniques n ire FIGURE 1 Results Separation of Labelled Glycans Based on Charge Size and Polarity The GlycanPac AXH 1 column can be used for qualitative quantitative and structural analysis as well as characterisation of uncharged neutral and charged glycans present in proteins Figure 1 shows bovine fetuin on the Glycan
16. Manual Exchange 0 5 Automated 0 5 50 minutes four spectra and their Recovery Time Wait for Purge 5 10 No Recovery Time 0 analyses aids Far IR Background Collect BKG 0 5 Collect BKG 1st 0 5 NON OOK mules and total hands on time was 45 Far IR Collect Load Sample 2 Load Sample 1 minutes representing inefficient Collect Spectrum Collect Spectrum m use of the analyst s time Change Optics NIR Manual Exchange 0 5 Automated 0 5 In contrast the results shown in Recovery Time Wait for Purge 5 No Recovery Time 0 Figure 5 emerged from a single Collect Background Collect BKG 0 5 Collect BKG 0 5 OMNIC macro operation The Collect Sample Load Sample 1 Collect SAM 0 5 macro was programmed to begin Collect SAM by collecting backgrounds for Data Analysis Search Perform Search 2 Automated Search 0 5 the mid and far IR ATR and Total Time 295345 6 5 then switched to the 1550 Raman module Next the samples were loaded the and Raman sampling stations After optimising the signal using the autofocus feature of the Haman module the macro was initiated and the analyst walked away From starting the macro to completion of the final report the analysis took less than 12 minutes representing a time savings of over 7096 The actual data collection time was again 5 minutes however total hands on time for the analyst was only 2 minutes a highly efficient use of the analyst s and the instrument s time Con
17. Sorvall superspeed centrifuges and rotors that can be used in concert with the commercially available DNA preparation kits and also provides a low cost method to obtain plasmid in large scale about 1 mg without using a kit Procedure PROTOCOL 1 Cost Efficient Large Scale DNA Plasmid Prep using only floor standing Superspeed Centrifuges from start to finish Hui Li Department of Pharmacology and Molecular Toxicology University of Massachusetts Medical Center Worcester MA This procedure describes the isolation of plasmid DNA using a floor model superspeed centrifuge from start to finish The Thermo Scientific Sorvall LYNX 6000 superspeed centrifuge employs a diverse array of rotors and can accommodate a wide variety of applications 1 Using the appropriate antibiotic selection grow 500 mL or more of bacterial culture usually overnight at 37 with shaking 2 Divide culture by pouring into 250 mL bottles Pellet bacteria by performing centrifugation in a Sorvall LYNX 6000 superspeed centrifuge with a Thermo Scientific Fiberlite carbon fiber rotor such as the Fiberlite F14 6x250y F12 6x500 LEX F10 4x1000 LEX or F9 6x1000 LEX rotor with the following parameters 6 000 x g for 15 min at 4 C 3 Decant supernatant and thoroughly resuspend bacteria in one of the bottles with 15 mL buffer A Transfer bacteria to the other bottle and resuspend the combined pellets There should be no clumps remaining
18. Touch Point to perform instrument setup and operation providing a touch and done workflow The removal of manual handling and exposure of the optics to the environment means instant readiness Second the user need no longer care about which optics are installed As seen in Table 1 the potential for error in manual operations is apparent when the array of possible component combinations is considered With the Nicolet 1550 spectrometer however a user simply presses the Touch Point on the instrument to automate the configuration and ready the instrument for the experiment For example pressing the Touch Point on the i850 module automates the setup without requiring any understanding of which optics are used What matters is performing NIR analysis not worrying about choosing the right components The instrument takes care of this step Integration of the spectrometer with its modules and components allows the user to expand capabilities increasing productivity with tools such as Upto three detectors such as near mid and far IR 1 550 Raman sample compartment module The built in diamond 1650 ATR sampling station The iS50 module with integrating sphere or fibre optics The iS50 GC IR module Asample compartment thermal gravimetric analysis IR TGA IR Interface Figure 1 describes the analytical power the user can achieve with the iS50 spectrometer to obtain a
19. Vantaa Finland gt ClipTip interlock technology the next generation for pipettes The most common type of pipettes in the lab are air displacement pipettes An airtight seal between the pipette and the tip is crucial for the pipette s functionality and any compromise on the sealing affects pipetting performance Traditionally air displacement pipettes have relied on friction between the pipette and the tip to form a seal The sealing is dependent on many factors including the tip altachment force used and might also be compromised through general use or when touching the vessel wall during pipetting With the new Thermo Scientific F1 Clip Pipetting System the attachment of tips is accomplished with an interlock technology between pipette and tip Breakthrough ClipTip Technology interlock technology utilises flexible clips positioned evenly around the top of the tip During attachment the unique tip fitting shape opens the clips allowing it to pass over the fitting flange and return to closed position The clips lock the tip behind the flange creating a complete seal with the sealing ring figure 1 In addition the lock ensures the tip will not loosen compromising the seal or even potentially fall off during routine pipetting or touch off 24 ejector Clip Opener Clip Tip fitting flange Tip fitting sealing ring Tip Figure 1 Clips closed behind the flange The t
20. and the mid IR background is collected in 1 5 minutes The sample is loaded and the spectra are collected in sequence All times are approximate Experiment Source Beamsplitter Detector Accessory Mid IR Transmission Thermo Scientific Polaris V KBr KBr DLaTGS standard Cells Far IR Transmission Polaris Solid Substrate Polyethylene DLaTGS Cells w Far IR Windows Near IR Transmission White Light CaF InGaAs Cuvettes Mid IR Polaris KBr Dedicated DLaTGS 1550 ATR Far IR ATR Polaris Solid Substrate Dedicated DLaTGS 1550 ATR FT Raman Raman Laser CaF Raman InGaAs 1550 Raman 2 no value to operations wasting the analyst s precious time Integration and automation on the spectrometer eliminate non productive wait times improving efficiency As an example Table 2 compares the steps needed to perform a full spectral analysis from far IR to near IR between the manual method Typical and the Nicolet iS50 method with built in iS50 ATR and 1550 NIR module This represents three spectral ranges in one sampling operation a unique power of the instrument Most important the built in 1550 ATR optics and detector permit spectral data collection in both the mid and far IR regions The analysis time decreases from around 30 minutes to less than seven With the Nicolet iS50 spectrometer the user is able to load two sampling locations the built in ATR and the Integrating Sphere module start the macro and walk away while in the manual ope
21. enjoyed reading Bio Innovation please pass it on to a colleague They may enjoy reading the magazine and could learn something that could move their research forward Sign up for Bio Innovation Ifyou are new to Bio Innovation and have received this copy from a colleague but would like your own why not register for your own copy email Bio Innovationgthermofisher com and guarantee that you are one of the first to receive the magazine when it is published Next PCR generation sing ery New Bainpro rw g 7 Stem 9 Ss existing digital PCR a Dye Un ath Achieve ur Rr Obtainin sults 1110 optimal and PCR io repro tions Monitor j Oring tos better m pD sede vtid NH Ec With Biolumine fey 25 in Groundbre The discovery biol M cardiovascular amp stroke al Publish Bio Innovation If you ve used our products in an application and you feel others would benefit from the information generated why not share it with the wider scientific community Send your ideas or abstracts to us at Bio Innovation thermofisher com Tell us what you think we would like to get your feedback on the content of the magazine so that we can improve it and increase your reading enjoyment Send l your comments or suggestions
22. exposure chemical concentration and centrifugal force Physical and chemical changes which may be caused by chemical exposure include Absorption of solvents resulting in softening or swelling of the plastic eGtress cracking of the plastic Permeation of solvent in the sample through the plastic Dissolution of polymer in the sample The Nunc conical centrifuge tubes made of premier quality polypropylene Table 3 displays information chemical compatibility of common reagents to polypropylene centrifuge tubes gt D Sample volume Before selecting a centrifuge tube size the desired sample volume should be determined The sample volume is limited to the available rotors in the laboratory and the protocol being used Generally a centrifuge tube should be filled at least 75 some cases Such as with ultracentrifuge tubes it is required that tubes are completely filled to prevent failure Using centrifuge _ tubes less than half filled to capacity can lead to high levels of material stress gap between the tube and the rotor good contact between the tube and the rotor where the g force applies gt Figure 1 Swing out bucket rotorand tube fit illustration Centrifugal forces are exerted on the conical and lower portion ofthe tube The imperfect fit of the lower portion of the tube to the rotor leaves a gap wh
23. for improvement to lea a 7 er 5 Bio Innovation thermofisher com If you missed out on receiving a previous edition of Bio Innovation request a copy today email bio Innovationethermofisher com the world leader in serving science we are uniquely positioned to combine our unrivalled depth of product application and service expertise with our extensive range of Scientific Healthcare Environmental and Industrial Process products to provide tailored solutions for each amp every customer Australia For customer service call 1300 735 29 Visit us online at www thermofisher com au 14 X Cg New Zealand For customer service call 0800 933 986 77 5 6 T E Visit us online at www thermofisher co nz Y pes 2013 Thermo Fisher Scientific Inc All rights reserved oet dv Eug Fii atu 8
24. heat and circulates water back to the water tower or chilled water reservoir system A graphic example of a water cooled condenser is shown in figure 1 Ultra low freezers using chilled water condensers must have water flowing through the condenser at all times Chilled water systems likely will require periodic maintenance The freezers can be protected by the connection to city or house water during the maintenance process The following procedure is recommended to switch between water used for cooling Connections must comply with local building codes The conversion process assumes chillers and house water are available Through water cooling the heat rejection rate of a ULT can be reduced by 70 to 8096 Water cooling condensers offer an alternative to tne extra heat load on HVAC system Water cooling reduces heat io the surroundings oy approximately 50 Process to switch from chilled water source to house water source Check water pressure in both systems Pressure value should be x value e Purge the house water line to remove air or particulates in the house water lines e Check the water pressure It must be at target pressure for house systems to protect the ultra low temperature freezers e Switch the valve to house water e off chilled water source e Monitor the temperature and water pressure on the house line imas Figure 2 Chilled water source vs house water s
25. instruments to consumables helping researchers identify biomarkers of disease so they can develop better treatments and one day find a cure would like to take this opportunity to thank you for choosing Thermo Fisher Scientific hope this edition of Bio Innovation provides you with some useful insight and assists you to achieve your results look forward to hearing directly from you and hope to meet you during my travels Tony Acciarito Director Scientific Business Thermo Fisher Scientific JNA 6 The proprietary MagJE T micro beads MagJET magnetic bead based technology utilises proprietary high capacity paramagnetic particles optimised to isolate nucleic acids with superior purity and yields compared to other kits on the market Due to large total surface area MagJET micro beads exhibit high binding capacity resulting in superior nucleic acid yields and recovery rates typically exceeding 8096 Uniform bead size and shape ensure robust performance and consistent results with less sample to sample variation The MagJET micro beads are highly stable in solution and respond to an applied magnetic field making the MagJET kits an ideal choice for high throughput automation Designed for great performance Magnetite content 60 High binding capacity Fast magnetic response Uniform size and shape Ideal for automation High recovery gt 80 Proprietary technology
26. labelled glycans especially higher sialic acid glycans However fluorescently labelled glycans generally provide better and more MS MS fragmentation peaks The GlycanPac AXH 1 column is useful for the analysis of both native and labelled N glycans depending on the amount of sample available If the amount of the sample is not extremely limited analysis of unlabelled glycans using the GlycanPac AXH 1 is highly feasible Structural Analysis of N Glycans from Antibodies by LC MS Using GlycanPac AXH 1 Column Antibody research has gained significant interest as a part of the development of protein biotherapeutics Glycosylation of antibodies is a prime source of product heterogeneity with respect to both structure and function Variation in 1 gim tart Frain bears 1 qe FIGURE 3 FIGURE 1 Separation of 2AB labelled N glycans from Bovine fetuin by charge size and polarity FIGURE 2 Comparison of 2AB labelled N glycans standards and 2AB N glycans from fetuin FIGURE 3 LC MS analysis of 2AB labelled N glycans from Bovine fetuin by the GlycanPac AXH 1 1 9 um column with MS detection FIGURE 4 LC MS analysis of 2AB labelled N glycans from Bovine fetuin by a commercial amide HILIC column 1 7 um with MS detection www thermofisher co nz bio innovation www thermofisher com au bio innovation FIGURE 5 LC MS analysis of nativ
27. need to be stored between 2 and 8 C and must not be exposed to freezing temperatures which will irreversibly reduce their titre However the development of the varicella vaccine 1995 and the more recent introduction of the live attenuated influenza vaccine LAIV have increased the complexity of vaccine storage These vaccines including Varicella Chickenpox and Zoster shingles as well as LAIV must be maintained in afrozen state 18 C is recommended without any freeze thaw cycles occurring 9 Temperature fluctuations can result from sample retrieval by multiple users unpredictable defrosting cycles or poor insulation Accidental freezing of freeze sensitive vaccines which represent over 31 of the vaccines on which UNICEF spent 318 million in 2005 9 can also irreparably damage the chemical structure of a vaccine rendering it ineffective In addition certain freeze sensitive vaccines contain an aluminum adjuvant that precipitates when frozen resulting in a loss of efficiency and potency To prevent temperature deviations modern clinical refrigeration equipment provides microprocessor controlled in built monitoring systems These can include a graphic thermometer that confirms normal high or low temperature conditions with the optional ability to automatically record temperatures over a period of time in a chart format Alternatively the placement of a US National Institute of Standards and Technolo
28. sensitivity fold concentration range Thus the samples can always be The Cypridina assay produces the most stable light emission measured without additional dilution steps and therefore it is recommended to integrate the signal somewhat longer than with assays with a more unstable signal Figure 3 illustrates the flash type reaction kinetics of Gaussia The Firefly flash reaction shows the most unstable kinetics Cypridina Renilla and Firefly luciferases the assays reach therefore the recommended integration time is the shortest the light emission maximum in a few seconds after the reaction A usable integration time range is a range where changing has been started Cypridina and Renilla assays show a fairly the integration time has only a minor effect on the assay stable light emission for several seconds and the Gaussia performance When using shallow well 384 plates with smaller Recommended Integration Times Normal 384 well plate Shallow well 384 well plate Luciferase Optimal time s Usable range 5 Optimal time s rUsable ange 5 gaussia 7 1 1 5 f 4 7 cypridina 5 1 5 7 3 green renilla 2 12 10 4 10 red Firefly 1 0 5 1 1 3 assay volumes it is recommended to use longer integration times than with normal 384 well plates with higher assay volumes See Table 1 Even though this application note only presents the results of assays performed in a 384 well plate format measuremen
29. 37 C for 1 hr 13 Precipitate the DNA for 30 min on ice with 0 5 volume 20 PEG 8000 2 5 M NaCl or with one volume of 1396 PEG 8000 1 6 M NaCl 14 Perform centrifugation in the Thermo Scientific Fiberlite F14 14x50cy rotor with 15 mL conical adapters or in the Thermo Scientific A21 24x15c rotor with the following parameters 20 000 for 20 min at 4 15 Carefully remove all the supernatant The pellet may be very translucent and smeared along the outer edge of the tube It is critical to remove all the PEG 16 Dissolve the DNA in 700 uL of TE or 10 mM Tris and transfer to a high performance 1 5 mL micro centrifuge tube 17 Add an equal volume 700 uL of phenol vortex 30 sec and perform centrifugation in the Thermo Scientific Fiberlite F2 48x1 5 rotor with the following parameters 19 000 x g for 5 min at 4 C 18 Remove the upper aqueous phase and transfer to another high performance microtube containing 350 pL phenol and 350 uL chloroform 19 Vortex and perform centrifugation with the following parameters 19 000 x g for 5 min at 4 C 20 Perform a final extraction in 700 uL chloroform Repeating the phenol chloroform extraction may increase DNA purity A back extraction once with 700 uL of TE can increase yield 21 Precipitate the DNA by the addition of 0 8 volume of isopropanol and 0 1 volume of NaOAc pH 4 5 5 5 Mix several times by inversion Perform centrifugation in the Fiberlite F27 48x1 5 rotor wit
30. 4652 cabs CITROEN S neededto ensure good fitting Legend 1XR TX 400 75003682 4696 ofthe tube to the rotor Legend 1XR TX 200 75003771 5580 Multifuge M X3R TX 750 75003639 4816 Multifuge X3R BlOShield 1000A 75003642 5000 Multifuge X3R Fiberlite F15 8x50cy 010 0378 06 24446 Multifuge X3R BlOLiner 75003673 2739 Evolution TM Fiberlite F13 14x50cy 010 0378 06 15000 Maximum speed for the centrifuge rotor system is reached Table 2 Recommended Maximum RCF for Thermo Scientific Nunc 50 mL Conical Tubes Catalogue 339652 339653 Thermo Scientific Centrifuge Model Thermo Scientific Rotor Model Thermo Scientific Adapter Catalogue Maximum RCF x g Legend 1XR BlOShield 720 75003677 6500 Legend 1XR Fiberlite F15 6x100y 75003103 23500 Legend 1XR TX 400 75003683 4696 Legend 1XR TX 200 75003803 5580 Multifuge X3R TX 750 75003638 4816 Multifuge X3R BlOShield 1000A 75003643 6500 Multifuge X3R Fiberlite F15 8x50cy not necessary 20000 Multifuge X3R BlOLiner 75003674 Evolution Fiberlite F13 14x50cy not necessary 15000 Maximum speed for the centrifuge rotor system is reached Summary Table 3 Chemical Compatibility of Common Reagents As in most endeavors gathering the to Polypropylene Centrifuge Tubes correct information ahead of time is essential In this study we demonstrated Phone Rating the important factors to consider Alcohols Butanol pure S when select
31. 5 seconds at a sampling rate of ten readings per second Each well was dispensed and measured before proceeding to the next well Luminescence signals were integrated from kinetic curves for different time periods Additionally individual signals and signal maxima were collected for comparison of different time points Assay sensitivity and dynamic range were calculated for each set di 2m uam Ng gt Figure 1 Optimal microplate readers for Pierce Reporter Assays Varioskan Flash on the right and Luminoskan Ascent on the left o nz bio innovation www thermofisher com au bio innovation EN www thermofisher c gt Figure 2 Typical standard Mab idi Gaussa luciferase assay in normal 884 well plate Cypralina luadarase assay iri well 384 plate luciferase flash assays with 150 different plate formats 90 84 2 24 24 ima oe 40 40 40 59 40 329 20 40 09 14 26 74 50 50 4 30 28 10 00 18 media Lag sfulwell Cypridina media gt Results assay shows about 3 signal decay per second after reaching The Pierce Luciferase Reporter Assays and the Varioskan maximum The firefly flash assay represents the fastest flash Flash provide very high performance and enable use of very reaction where the signal decays a
32. GF1 are plotted against the calculate the concentrations of on the samples area ratios Replicate analyses of a single plasma donor demonstrated CV of 8 596 Thermo Scientific Protein A G MSIA Tips Thermo Scientific Versette Liquid Handling Platform e Anti human IGF1 antibody Humanrecombinant IGF1 IGF1 standard calibration cuve was generated Recombinant LR3 IGF1 Internal reference standard Thermo Scientific Pierce BupH Phosphate Buffered Saline PBS Antibody dilution buffer 10mM MES 0 1 polysorbate 20 pH 5 e EDTA plasma human donor IGF1 concentrations and usedto e rypsin IGF1 in unknown samples based e LC MS grade water e Thermo Scientific Optima grade Formic Acid Thermo Scientific Optima grade Acetonitrile ACN Standards dilution buffer 10 g L BSA in PBS pH 7 2 40 uL plasma IGF1 standards with 20 uL 0 5 mg L internal reference standard in standards dilution buffer followed by 100 uL PBS 0 396SDS and incubated at room temperature for 30 minutes prior to extraction and enrichment with Protein A G MSIA Tips Antibody Loading of Protein A G MSIA Tips Protein A G MSIA Tips were loaded with 100 uL rabbit anti human IGF1 antibody 0 01 mg mL following protocols provided in the user manual total processing time 30 minutes Extraction and Enrichment Co extraction and enrichment of IGF1 and LR3 IGF1 were pe
33. H 1 column 1 9 um with fluorescence detection The relative amount of AH 1 Es rrr ums rtu mo Eira Pe ee Li Vita y svn un E F d cia 1 LE each charge state curve A standards cur chromatographic analysis of with the injection of differen 0 1 to 5 pmole as estimated using a standard drawn using the data from the labelled A2 glycan standard unt of samples starting from Conclusion The GlycanPac AXH 1 column separates glycans with unique selectivity based on charge size and polarity not possible with commercial HILIC columns LC ESI FTMS or FI MS MS analysis of both native and labelled glycans from proteins and antibodies were carried out successfully using GlycanPac AXH 1 columns The GlycanPac AXH 1 column is useful for both high resolution charge based separation and easy quantification of glycans The GlycanPac AXH 1 columns are compatible with various MS instruments These new columns have high chromatographic efficiency and excellent column stability The GlycanPac AXH 1 column is also useful for the separation of reduced O glycans from proteins and mucins The GlycanPac AXH 1 column is useful for the analysis of charged and neutral glycosylaminoglycans and glycolipids m Do eo Moa 1 cr
34. MPEST Figure 5 Kinase A enzyme titration at 15uM ATP TEMPEST Figure 6 Inhibitor A at 15 and 250 uMATP Figure 7 Inhibitor B at 15 and 250 uMATP Figure 8 Competitive inhibition of Kinase A by Inhibitor A Figure 9 Competitive inhibition of Kinase A by Inhibitor B Figure 10 A375 cells dispensed manually Figure 11 A375 cells dispensed using Tempest Table 1 1 50 values of both inhibitors at various ATP concentrations Thermo Scientific M Barnstead M Type 1 water purification systems Why is UV intensity monitoring important for ultrapure water UV intensity monitoring is an innovative technology designed to ensure that the total organic carbon TOC reading is accurate providing superb reliability for ultrapure water The monitoring and display of the TOC content in product water has become increasingly important as biochemical methods become more sensitive to low levels of organics In addition to visualising the resistivity of the ultrapure water you need to quantify the amount of organic impurities in the water Organic free water is critical to applications that are sensitive to organics such as HPLC and GC MS It is imperative that the measurement be monitored for accuracy to prevent negative results TOC monitoring is a useful technology that provides a real time measurement of the actual level of organics in the product water Product water is regularly being sampled and tested for the leve
35. Pac AXH 1 1 9 um 2 1 x 150 mm column using fluorescence detection The separation and elution of glycans are based on charge the neutral glycans elute first followed by the separation of acidic 2AB labelled N glycans from monosialylated disialylated trisialylated tetrasialylated and finally pentasialylated species Glycans of each charge state are further separated based on their size and polarity The retention time of each glycan charge state was confirmed using 2AB labelled glycan standards as shown in Figure 2 Separation of glycans is based on charge size and polarity which provides significant structural and quantitative information The chromatographic profiles shown in Figures 1 and 2 detected by fluorescence detection provide qualitative information about the separation of N glycans The structure of glycans present in each peak was determined from the LC MS study using the GlycanPac AXH 1 1 9 um column as shown in the following section LC MS and LC MS MS Analysis of 2AB Labelled N Glycan Using GlycanPac AXH 1 Column The coupling of the GlycanPac AXH 1 column to MS was also explored This is particularly attractive as MS with it s ability to provide structural information enables in depth analysis of complex glycans 2AB labelled N glycans from bovine fetuin were separated on the GlycanPac AXH 1 column and analysed Exactive mass spectrometer Data dependant MS MS spectra were acquired on all precursor io
36. X qPCR MasterMix NTC is the no template control ww C rot s 08 10 R 0 9984 res 4 E 4 bbb tna 1 1 1 Human c Myc gene was amplified with the Luminaris Color HiGreen qPCR Master Mix on the PikoReal 96 Real Time PCR System using reverse transcribed from HeLa cells total RNA 10 ng 0 1 pg by Maxima First Strand cDNA Synthesis Kit for RT qPCR Cat K1641 The insert shows the melting curve analysis results indicating high specificity of the amplification Reaction efficiency E 98 196 R 0 9984 Human c Myc gene was amplified in 96 individual reactions from 10 ng of human genomic DNA with the Luminaris Color HiGreen Low ROX qPCR Master Mix on the ABI 7500 Real Time PCR instrument Calculated CV 0 24 Luminaris Color qPCR Master Mixes 2X contain a blue dye The 40 Yellow Sample Buffer is included as a separate vial This buffer can be mixed with the samples to provide visual aid when pipetting samples 6086 86868 29 0099 ose White reaction vessels are especially good for qPCR applications as they deliver higher signal intensities than clear vessels However traditional colourless reaction components are poorly visible in white wells making reaction setup more difficult Luminaris Color qPCR Mast
37. all performance the internal capacity of 2 inch vials over previous generation freezers 1001605 ognence gaining up to 696 more capacity in the same footprint The innovative dud ae es UC Veg xr us touch screen user interface allows monitoring of the freezer s health DC 24 7 and access a detailed event log simply touch the heart icon on the pion exch ID natant ret EST main screen to see freezer s temperature access settings and operating epe ure logo parameters Additional security options can be added with swip card cs es time and increase rotor security user access to enable full users monitoring y NanoDrop Lite The Thermo Scientific NanoDrop Lite Soectrophotometer is a compact personal UV Vis microvolume spectrophotometer that complements the full featured NanoDrop 2000 2000c and NanoDrop 8000 instruments The NanoDrop Lite provides rapid accurate and reproducible microvolume measurements without the need for dilutions It uses the same sample retention system that has become a hallmark of NanoDrop instruments the sample is held in place by surface tension only The NanoDrop Lite performs basic microvolume measurements Its compact design with built in controls and software make the NanoDrop Lite small enought to fit on any benchtop The patented sample retention system allows sample
38. ases Thus for materials like organometallics metal oxides the IR absorption shifts below 400 cm and below the range of Standard KBr optics Numerous polymers sugars and other large molecules also have far IR information which may be useful or definitive to the analyst Traditionally collecting FT IR Spectra in both the mid IR and far IR region entailed significant Sample preparation This included changing hygroscopic optics and multiple detectors and risking altered system performance from water vapor The Nicolet 1650 spectrometer enables rapid analysis over the full mid IR and well into the far IR region 4 000 cm to 80 when equipped with the 1550 1550 ATR and the correct beamsplitters The typical multi range FT IR application requires opening the spectrometer to swap beamsplitters This requires care in components Unlike most spectrometers operating the Nicolet l handling costly components and exposes the internal optics to the environment by disrupting purge or desiccation This activity adds recovery period to re equilibrate the instrument before quality data can be collected These wait times add Table 1 Experiments made possible with the Nicolet i550 FT IR Spectrometer Table 2 Far infrared analysis Typical versus Nicolet i550 process With the 1650 ATR present the far IR background BKG is collected the i550 ABX swaps beamsplitters
39. ates by the manufacturers Phusion High Fidelity DNA Polymerases performed the amplification in less time and with fewer units of enzyme than the other polymerases Reliable amplification of human mitochondrial DNA can be accomplished more quickly and cost effectively with Phusion High Fidelity DNA Polymerases Bio Innovation Issue 7 Introduction Human mitochondrial DNA is a 16 6 kb circular double stranded molecule Mutations deletions duplications and point mutations in the mitochondrial genome leading to mitochondrial dysfunction are increasingly recognised as a contributor to a wide range of human diseases Mitochondrial dysfunction is involved in diseases such as diabetes cancer heart diseases and migraine In addition neurodegenerative are associated with mitochondrial dysfunction Amplifying the whole mitochondrial genome using PCR has been found to be an efficient method for detecting mitochondrial DNA deletions involved in human diseases Amplifying the whole mitochondrial genome is useful for detecting relatively large mitochondrial deletions for other mutations e g point mutations shorter target fragments are usually amplified Materials and Methods Phusion High Fidelity DNA Polymerases consist of a Pyrococcus like enzyme with a double stranded DNA binding domain which gives the fusion polymerase high processivity and fidelity The performance of Phusion High Fidelity DNA Polymerases and three polymerase
40. both instruments can equipped with optical filters for measuring dual luciferase assays The filters have been specifically optimised to separate the two luciferase signals as efficiently as possible for the Pierce Dual Luciferase Assays and therefore they ensure excellent performance gt Materials and Methods The study of this application note focuses on performing flash type Luciferase Reporter Assays low assay volumes with normal 384 and shallow well 384 plates Instrument Varioskan Flash multimode microplate reader equipped with a luminometric measurement module and an onboard dispenser for automatic reagent addition Kits Flash type Luciferase Reporter Assay Kits for Gaussia dispensing and measurement from sample to sample Pipetting Microplates white 384 well OptiPlate Perkin Elmer Cypridina Renilla and Firefly luciferases Cat No 6007299 and white shallow well 384 well plate Thermo Scientific NUNC Cat No 264706 Luciferase samples were diluted in the Cell Lysis buffer provided in the kit to create a concentration series of over eight orders of magnitude Aliquots of 4 ul normal 384 well plate or 1 ul shallow well 384 plate of each dilution were added into the microplate wells Instrument control software was programmed to 20 ul or 5 ul of luciferase assay reagent with a dispenser and to measure the signal kinetically for 1
41. bout 20 per second during _ low assay volumes with normal 384 well and shallow well the first couple of seconds 384 well plates All luciferase reporter assays show perfect linear response over wide concentration range with both Using automatic dispensers in the luminometer for reagent normal and shallow well plates Real assay dynamics reaches addition is highly recommended to ensure exact timing in the eight orders of magnitude with the Gaussia and Cypridina measurements especially in Gaussia and Firefly assays If the flash assays Figure 2 The Renilla flash assay also shows a luminescence reaction is initiated by manual pipetting it may remarkably large dynamic measurement range Flash assay lead samples to be measured in different time points of the with Red Firefly luciferase shows a somewhat reduced dynamic kinetic reaction thus increasing sample to sample variations range about 5 to 6 orders of magnitude The Flash can automatically adjust the photomultiplier gain to increase Assay sensitivity is dependent on the measurement integration the assay dynamic range to eight orders of magnitude time but its effect is not very strong About a one second This unique feature means that the luciferase samples measurement time provides good sensitivity but a few have any luciferase concentration over a large 100 million seconds more be required for the best possible
42. ca Wang and Patrick K Bennett from Thermo Fisher Scientific for their help and permission to use their UHPLC and MS instruments glycosylation is one of the main factors in product batch to batch variation 23 affecting product stability in vivo and significantly influencing Fc effector functions in vivo A representative example of the chromatographic separation of antibody glycans is shown in Figure 6 where 2AA labelled N glycans from IgG were separated using the GlycanPac AXH 1 column 1 9 um Characterisation of glycans in each peak was performed by LC MS MS and results are shown in Figure 7 Three different glycan charge states were found in this human IgG the majority of glycans are neutral or monosialylated with minor amounts of disialylated glycans Separation of glycans based on charge provides advantages compared to other commercially available HILIC columns Quantitative Determination of Glycans Based on Charge Quantitative analysis of each glycan is essential for quick assessment of glycan variation in protein batch comparisons and for comparison of diseased to normal cell glycosylation profiles In addition quantitative analysis of glycans separated based on charge state also provide a tool for calculating the relative amounts of different sialic acid linkages after enzymatic digestion with silidase S and sialidase A Figure 8 shows the quantitative analysis of 2AB labelled N glycans based on charge the using GlycanPac AX
43. cess scale up options for a new product launch or troubleshooting customer complaints Such diversity of applications requires the selection and installation of the correct instrument accessory as well as choosing the optimal source beamsplitter detector optical path and experimental conditions Manually changing components and sampling parameters requires skill and may risk exposure of expensive optics to the external environment dust fingerprints or water vapor In addition changing these parameters can result in extensive wait times to equilibrate the instrument before the next measurement These manual reconfigurations provide little added value to the laboratory workflow Users must plan and set up batch experiments to minimise the number of steps This creates bottlenecks limiting access to the full capability of the instrument As a result labs are less able to address emergency situations without interrupting the batch run and resetting the instrument parameters For instance analysis of a polymer with additives requires mid IR and far IR plus Raman spectroscopy This would entail three beamsplitter changes with associated risks in handling expensive components and instrument recovery times between changes The productivity improvements with the Nicolet i850 FT IR spectrometer come from two main sources First the internally mounted 1550 ABX Automated Beamsplitter Exchanger uses one button simplicity described as a
44. ciferase assays Glow type luciferase reporter gene assays produce a very stable luminescence light signal that lasts for approximately one hour As the light emission decays slowly measurement does not have to be performed immediately after the addition of substrate The stability of the light makes it possible to pipette all the assay reagents manually without need of reagent dispensers installed in the instrument Glow type assays are generally less sensitive than flash type assays 2 Flash type luciferase assays Flash type luciferase reporter gene assays are in general more sensitive than glow type assays Luminescence signals are transient in flash type assays so the signal peak is reached soon after assay reagent addition Therefore flash type assays often require the assay reagent to be added with automatic dispensers installed in the luminometer Reagent dispensers in the instrument facilitate signal monitoring right from the start of the luminescence reaction and enable precise timing between the assay reagent manually increases sample to sample signal level variations especially in flash type assays and miss the capture of true flash signal due to delay in measuring the signal after manual reagent addition 3 Dual luciferase assays Thermo Scientific Dual luciferase assays are highly sensitive flash type assays for multiplexing The one step assays are based on having two luciferases emitting luminescent light at spectrally dis
45. clusion Many forces contribute to new pressures on industrial analytical laboratories increased sample loads decreased staffing retirement of experts and shrinking budgets The Thermo Scientific Nicolet i850 FT IR spectrometer makes significant contribution to alleviating these challenges through automation in a multi tasking single platform instrument Nicolet 1650 spectrometer greatly simplifies and streamlines workflows by decreasing the number of steps with one button ease and macro operations performed by the analyst In addition risks inherent in manual operations e g user error environmental exposure and long recovery times are eliminated Analysts of any skill level can successfully obtain meaningful results with minimal hands on time Technology designed to improve workflow can be found in the 1550 ABX and task specific modules i e Raman etc The Touch Point operation simplifies access to the full range of capabilities by automatically configuring the optics near mid and far IR and switching between sampling stations modules in seconds for enhanced productivity For the modern industrial lab the Nicolet i850 FT IR spectrometer offers a powerful new tool that goes beyond routine FT IR to more comprehensive analyses e g FI Raman and far IR adding value to laboratory activities in a compact easy to operate platform Figure 3 eg a Fa R Acetyiferrseane en 1550 ATR LL
46. cost effective transition from method development to high throughput protein quantitation The tags consist of TMTO zero the TMTduplex and the TMTsixplex set The Label Reagent allows testing and optimisation of sample preparation labelling fractionation and MS fragmentation for peptide identification and reporter detection without using the more costly isotope labelled compounds The TMTduplex allows duplex protein profiling for small studies The TMTsixplex allows sixplex protein profiling for multiple conditions including time courses dose responses replicates or multiple sample comparisons Each isobaric tag is based on the same chemical structure eliminating the need to modify labelling conditions or HPLC separation conditions between experiments Luminaris qPCR Master Mixes The Luminaris qPCR Master Mixes are specially formulated to produce the most consistent and reproducible qPCR data using probe SYBR Green chemistry across all real time PCR platforms The Master Mixes contain Thermo Scientific Hot Start Tag DNA polymerase in an optimised reaction buffer for increased reaction efficiency specificity and sensitivity Uracil DNA Glycosylase UDG included in the Master Mixes degrades the uracil containing PCR products carried over from previous experiments membrane provide the last line of defense against cell culture contamination Now you Complet Filter Units Now Stem Cell Tested Thermo Scient
47. different enzymes With both formats we found the CV s for 0 366 0 003 0 755 Kinase R 1 584 the whole plate to be within the acceptable range of 5 0 388 0 004 1 011 Kinase 5 1 721 0 386 0 003 0 866 Kinase V 2 085 Workflow comparison Our current workflow for establishing assay parameters involves 3 separate steps a Titration to determine the enzyme concentration for a 3096 conversion Fig 1 b Kinetic run to determine the ATP Km of the enzyme Fig 2 C Final titration to confirm the established parameters Fig 3 These resource intensive steps can be eliminated with the Tempest which by virtue of its independent channel control allows a factorial dispense of ATP and enzyme The resulting matrix design enables us to attain the assay parameters in a single step Fig 4 5 Figure 1 Figure 2 VMAX 1 296 KM 16 89 Conversion 0 5 1 0 1 5 2 0 2 5 Enzyme conc ng ul Figure 3 Figure 4 VMAX 0 7459 KM 15 06 Conversion 0 2 0 4 0 6 Enzyme conc ng ul Figure 5 Conversion conc of Enz ng ul Figure 10 Figure 11 Summary Modality of inhibition We evaluated the Tempest s efficiency in modality of inhibition experiments By dispensing a gradient of ATP down the plate we studied the response of two in house competitive inhibitors Data Figures 6 amp 7 show IC50 curves for inhibitors A 8 B at distinct ATP concentrations IC50 values at o
48. e in combination with the specially optimised reaction buffer considerably reduces non specific amplification and formation of primer dimers The highly reproducible qPCR results achieved from a wide range of input DNA amounts allow accurate quantification of genomic plasma viral and cDNA templates A m Color for control over pipetting process Luminaris Color qPCR Master Mixes incorporate an inert blue dye which does not affect the qPCR reaction but significantly enhances the contrast between the reagent and plastic The Yellow Sample Buffer complements the colour system by making the overall dispensing of qPCR reaction components quick and easy This is particularly important for users of opaque white reaction plates and tubes generally recognised as giving superior QPCR results ole 100 1 R 0 9998 ie Wide dynamic range 10 to 10 copies of plasmid DNA were amplified using the Luminaris Color Probe Master Mix on the PikoReal 96 Real Time PCR System NTC is the no template control UDG effectively removes carry over contamination in qPCR Amplification of PGK 1 gene was performed from 100 ng 10 pg of human genomic DNA spiked with dU containing PCR product 3000 copies sample DNA was amplified with or without UDG treatment step on ABI StepOnePlus Real time PCR instrument using Luminaris Color Probe High RO
49. e N glycans from Bovine fetuin All the peaks are detected by MS detection in negative ion mode FIGURE 6 Analysis of 2AA labelled N glycans from human IgG FIGURE 7 Mass spectroscopic characterisation of glycans in each Figure 6 peak FIGURE 8 Quantitative estimation of each charge state glycan in 2AB labelled N glycan from Fetuin References 1 Varki A Biological Roles of Oligosaccharides All the Theories Are Correct Glycobiology 1993 3 97 130 2 Bertozzi C R Freeze H H Varki A Esko J D Glycans in Biotechnology and the Pharmaceutical Industry Essentials of Glycobiology Second Edition Cold Spring Harbor Laboratory Press New York 2009 Chapter 51 3 Guidance for Industry Scientific Considerations in Demonstrating Biosimilarity to a Reference Product Draft Guidance U S Department of Health and Human Services Food and Drug Administration February 2012 Online www fda gov downloads Drugs GuidanceCompliance RegulatoryInformation Guidances UCM291 128 pdf accessed Jan 18 2013 4 Bigge J C etal Non Selective and Efficient Fluroscent Labeling of Glycans Using 2 Amino Benzamide and Anthranilic Acid Anal Biochem 1995 230 229 238 5 Apte A Meitei N S Bioinformatics in Glycomics Glycan Characterization with Mass Spectrometric Data Using SimGlycan Methods Mol Biol 2010 600 269 81 Acknowledgements We would like to thank Mark Tracy Yoginder Singh Jessi
50. ed using the Sorvall LYNX 6000 superspeed centrifuge Centrifugation at 6 000 x g for 10 15 min at 4 C is sufficient to pellet bacteria grown 500 mL or larger culture For smaller volume cultures the time and speed for pelleting is reduced centrifugation at 5 000 x g for 10 min at 4 C is sufficient for a 150 mL bacterial culture Table 2 lists a selection of rotors the Sorvall LYNX 6000 superspeed centrifuge that will serve to accommodate small and large scale pelleting up to 6L needs prior to DNA purification Table 2 Thermo Capacity Speed Scientific Rotor place x mL rpm X g Fiberlite F9 6x1000 LEX 6x1000 9 000 17 568 Fiberlite F10 4x1000 LEX 4x 1000 10 500 20 584 Fiberlite F12 6x500 LEX 6x500 12 000 24 471 6x250 14 000 Fiberlite F14 6x250 30 240 BIOFlex HC 4x 1000 5 500 7 068 BIOFlex HC with adapter 4x500 5 500 7 068 BIOFlex HC with adapter 8x250 5 500 7 068 BIOFlex HS 4x400 7 000 10 025 Following the initial pelleting of bacterial culture DNA purification steps require centrifugation at gt 15 000 x g For ease of use and to ensure the integrity of samples the use of sterile 15 mL to 50 mL conical tubes is desired however these conical tubes are typically limited to lt 7 000 x g The unique Thermo Scientific conical tube rotors and conical tube adapters allow for the use of disposable sterile conical tubes at RCFs up to 63 409 x g Table 3 lists a selec
51. er Mixes overcome this difficulty and decrease the risk of pipetting errors In the image the blue wells contain only the 2X Master Mix The green colour indicates the sample DNA with yellow colour has also been added into the reaction References 1 M C Longo etal Use of uracil DNA glycosylase to control carryover contamination in polymerase chain reactionsGene 93 125 128 1990 P fv 2 ie d 2 Gee Er ng i am MI 1 y x L x F 1 M tip C 4 mili ME LX i IP m p 1 TAM B nmm Tj i 1 i a NUS WE ndr EE mam rre wer 1 T rar 1 4 1 4 1 x m M 1 bi i 4 d i a z 774 he d j i 4 f I i a E i j _ I 4 Li 1 i me X 1 1 ES s f i t gt x Am A 4 4 1 Chl m s J EET LL 3 XI It meu bois Wet Bele 5 E 79 t A Amy E ru 7 I hr Lt nsus o WA rae uu oe TE wo x e AD s E ER T MRNA m a Lar So PEN
52. gy NIST certified thermometer in the centre of the storage unit adjacent to the vaccine allows the unit s temperature to be read and documented twice a day Records are then kept for a minimum of three years as recommended in the US CDC guidelines 4 lf temperatures are found to be outside the recommended range action must be taken immediately Storage equipment that features an automatic defrost system is highly recommended as it prevents water ice frost or coolant leaks that could potentially harm vaccine samples Refrigeration units provide efficient compression technology and forced air circulation to maintain temperature uniformity throughout the cabinet They also provide a tight and secure door closure through the use of spring loaded self closing doors and effective door seals to offer additional vaccine protection Some units also supply Door Ajar alarms that notify users when the cabinet door is not closed properly Blown in insulation that conforms to the unit s shape prevents cold air escaping as well as internal temperature fluctuations due to exposure to ambient conditions Many modern laboratory refrigerators and freezers also supply audio and visual alarms that alert users to temperature deviations providing a real time warning that cannot be ignored when for example preventative maintenance is required Every clinic should be prepared with a recovery plan in case of problems that
53. h minimal user interaction The resulting IGF1 MSIA SRM using Protein A G MSIA Tips demonstrated a wide linear dynamic range Figure 2 and the ability to reproducibly quantify unknown IGF1 concentrations from plasma samples Table Loaded with IGF1 antibody the Protein A G MSIA Tips enabled for the efficient capture and enrichment of femtomole amounts IGF1 Table A with no additional sample preparation or depletion Figure 2 Protein A G MSIA Tips GFI MSIA SRM 055539 Perri OR eee 158 E ippeg itj IGF1 Reproducibility LLOD Assay Range n 12 0 nz 1 1500 ng mL 5 2 7800 femtomole 1 ng mL 5 2 femtomole 114 8 5 Amounts based on a 40uL plasma sample volume TableA Protein A G MSIA Tips IGF1 SRM assay characteristics Conclusion We described the development of a novel model system targeting human IGF1 to test the performance of Protein A G MSIA Tips Using this test system the MSIA Tips demonstrated the ability to reproducibly perform quantitative measurements using SRM detection These results clearly show how these devices can provided mechanism for the highly specific and reproducible enrichment of a target analyte from plasma Not only does this described SRM assay provide a uniform approach to technology evaluation it also serves asa template for the development of future LC MS MS based MSIA
54. h the following parameters 19 000 x g 15 min at 4 C 22 Decant supernatant and carefully wash the DNA pellet with 1 mL of 7096 EtOH 23 Air dry pellet and dissolve in 500 1000 uL Tris or TE 24 Measure the OD260 and OD260 280 and calculate DNA concentration DNA concentration in g L OD260 50 Dilution and assess purity Note An OD260 280 below 1 8 suggests impurities Double phenol chloroform extraction can improve the ratio Table 1 Solutions Needed Buffer A 50 mM Tris HCl pH 8 0 10 mM EDTA Buffer B 0 2 NaOH 1 SDS Buffer C 3 M Potassium Acetate pH 5 5 Isopropanol 70 Ethanol RNAase A stock solution 10 mg mL PEG NaCl solution 20 PEG 8000 2 5 M NaCl or 13 PEG 8000 1 6 M NaC Phenol Iris buffered to pH 8 0 Chloroform 10 mM Tris or TE 3 M Sodium acetate pH 4 5 5 5 PROTOCOL 2 Using Superspeed Centrifuges for Plasmid DNA Preparation with Commercially Available Kits Protocol 1 describes the process for plasmid DNA preparation without using a commercially available kit Many commercially available kits for plasmid DNA preparation that call for the use of a centrifuge with high speed and large volume capabilities such as the Sorvall LYNX 6000 superspeed centrifuge The initial steps in DNA preparation involves the pelleting of bacteria in which the plasmid of interest has been propagated Individual bacterial cultures with volumes up to 6 L can be process
55. ich may cause tube damage Upper portion Lower portion Conical bottom portion Figure 2 Fixed angle rotor and tube fit illustration Centrifugal forces are exerted on the sides of the tube and transferred to the rotor adapter spun at much higher RCF in comparison and can result in tube failure If the to a swing out bucket rotor available sample volume is smaller than the available rotor capacity adapters are available for most rotors from the manufacturer Table 1 Table 2 when evaluating centrifuge tubes it Adapters allow smaller volume tube is important to make sure that the to be used at the appropriate fill volume sample components will not harm the This is essential when the recommended plastic tube material Chemicals can maximum RCF is going to be applied for affect the strength flexibility surface centrifugation gt C Chemical compatibility In addition to the maximum RCF Table 1 and Table 2 display m the recommended maximum Table 1 Recomended Maximum RCF for Thermo Scientific M Nunc 15 mL Conical Tubes Catalogue 339650 339651 Nunc 15 mL and 50 mL Thermo Scientific Centrifuge Model Thermo Scientific Rotor Model Thermo Scientific Adapter Catalogue Maximum RCF x g conical tubes based on tests of 9 models of rotors in 3 Legend 1XR BlOShield 720 75003678 3500 types of centrifuge units In Legend 1XR Fiberlite F15 6x100y 75003095 2
56. ient A water 0 1 formic acid B acetonitrile 0 1 formic acid at 300 nL min over 60 min A Thermo Scientific Orbitrap XL ETD linear ion trap mass spectrometer was used to detect peptides using a top 3 CID 3 HCD experiment for peptide identification and reporter ion quantitation FIGURE 1 iodoTMT reagents and labeling reaction Mechanism of iodoTMT reagent reaction with cysteine containing proteins or peptides B Structure of iodoTMTsixplex reagents for cysteine labeling enrichment and isobaric MS quantitation Figure 1 FIGURE 2 Schematic of iodoTMTsixplex reagent workflow Six different sample conditions can be prepared for iodoT MT reagent labeling Labelled proteins are combined before iodoTMT peptide enrichment using immobilised anti TMT antibody resin and subsequent LC MS MS analysis of isobaric reporter ions FIGURE iodoTMT reagent labeling specificity and efficiency A Reduced BSA 100 ug was labelled with increasing concentrations of iodoTMT reagent lodoTMT reagent labeling efficiency was determined by peptide signal XIC of modified of cysteines compared to total cysteine containing peptides signal B Reduced BSA 100 ug was labelled with 10 mM iodoTMT reagent lodoTMT reagent labeling specificity was determined by comparing modified peptide signal to total peptide signal for different amino acids Labeling specificity and efficiency were also assessed by peptide spectral c
57. ientific Accela pump a PAL auto sampler and a Thermo Scientific lon Max source equipped with a high flow metal needle A mass window of full width at half maximum of 0 7 unit resolution was used in the SHM assays because immuno enriched samples have a very high signal to noise ratio Reversed phase separations were carried out on a Hypersil GOLD C18 column 50 mm x 2 1 mm 1 9 um particle size with a flow rate of 240 uL minute Solvent A was 0 2 formic acid and solvent B was 0 2 formic acid in acetonitrile and Discussion A model system for the Protein A G MSIA Tips based on Insulin Like Growth Factor 1 IGF1 was developed to serve as a template for future LC MS MS methods that perform quantitative immuno affinity proteomics The Protein A G MSIA Tip workflow Figure 1 proved to be simple and fast requiring as little as 1 hour to provide immuno purified IGF1 Figure 1 STEP STEP i STEP Incubation T 4 Antibody Incubation STEP j 9 amp Protein A G Dispense Eluate into microplate dry down 1 Buffer 2 Water Antibody Solution Analytical Sample LC MS MS IIS E y Reconstitution Reduction Alkylation Tryptic digestion 2h 50 C Using the Versette liquid handler equipped with these tips up to 96 samples were able to be processed in parallel wit
58. ific Nalgene Rapid Flow disposable Filter Units with PES polyethersulfone can guard your precious samples against contamination and safely culture embryonic stem cells using filtered media as long as you have the right filters and membranes Our recent stu ly Culturing embryonic stem cells using media filtered with Thermo Scientific MUR at i ua PES filter that Embryonic Stem Cells grown in media filtered through Thermo Scientific Nalgene Rapid Flow PES filters maintain normal growth acy all hout iud In media components or adding deleterious ing he filtration pro 5655 Thermo Scientific Water Thermo Scientific Water is a complete line of water purification technologies which includes solutions for your most critical and everyday application needs from electrodeionisation to reverse osmosis and distillation Our water purification portfolio features advanced ergonomics and technology including remote dispensing UV intensity monitoring small footprints and flexible dispensing options to provide a configuration that best suits your lab Many water systems can be easily upgraded to allow for additional capacity Nicolet FTIR Aa The Thermo Scientific Nicolet 1550 featuring purpose built accessories and integrated software is an all in one materials analysis workstation designed to help solve analytical challenges with The highly flexible system can upg
59. includes a refrigerator with a back up generator in the event of for example a power cut or natural disaster Today s refrigeration equipment can provide a full alarm function in case of power failure and a low battery alarm that displays when the alarm system battery backup is low gt Best practice vaccine protection The storage units selected should be of a high enough quality to negate the need for frequent maintenance and repairs which can compromise vaccine quality due to cabinet downtime and the transfer of samples to another location Temperature should be recorded twice daily and any deviations from the optimum range should be reported Studies have demonstrated that educating at least one staff member about correct monitoring and reporting of the refrigerator temperature significantly improves the maintenance of storage conditions with fewer deviations from optimal temperature ranges going unreported 8 It is recommended that clinics should use dedicated refrigerators and freezers to store vaccines Although combined refrigerator freezer units are acceptable for vaccine storage if each compartment has a separate door inconsistencies in temperature uniformity can arise making combined units less suitable for the storage of any temperature critical samples Clinics need to make allowances for the largest possible batch of vaccines that they will need to st
60. ing the conical tubes for Ethanol 100 centrifugation so that sample leakage or Isopropanol 100 S lossis prevented Choosing the correct _ centrifuge consumables alsoensures 5 proper execution of centrifugation and Methanol 9896 x 750 51 reduces the risk for potential damage to Propanol 100 S the centrifuge and rotor Cryopreservation Agents Dimethyl sulfoxide DMSO pure S The factors we discussed in this article Glycerol S include the RCF required by the protocol Detergents Sodium Dodecyl Sulfate SDS pure S the fit of the particular conical tube Triton X 100 pure inthe rotor sample volume and the Tween 20 5 chemical compatibility of the sampleto _ the tube material Users should verify the Hates Acetone 90 5 maximum RCF ofthe centrifugetubeto Acetone pure M ensure that the required speed does not Formaldehyde 10 51 exceed the manufacturer s rating Here Formaldehyde 30 M we recommended the maximum RCF i ofthe Nunc 15mLand50mLconical 120 51 tubes in several Thermo Scientific Formalin 3096 M centrifuge and rotor systems while taking Gluteraldehyde pure these factors into consideration Paraformaldehyde pure M Key Table 3 Other Beta Mercaptoethanol pure culture media and sera 5 gt References S Satisfactory xem pure S1 Satisfactory may cause discolou
61. innovation 1 A GOCI I tile Ac for cysteine peptide modification enrichment amp quantitation Ryan D Bomgarden Thermo Fisher Scientific Rockford IL USA Rosa Viner Thermo Fisher Scientific San Jose CA USA Karsten Kuhn Proteome Sciences Frankfurt Germany lan Pike Proteome Sciences Frankfurt Germany John C Rogers Thermo Fisher Scientific Rockford IL USA Overview Purpose Methods To develop an iodoacetyl Tandem Mass lag iodo T MT Reduced sulfhydryls of protein cysteines were labelled with iodo TM Tzero and or reagent for irreversible cysteine peptide labeling enrichment iodo I MTsixplex reagents Labelled peptides were enriched using an immobilised anti TMT and multiplexed quantitation antibody resin before mass spectrometry MS analysis Results We developed an iodo T MT reagent set to perform duplex isotopic or sixplex isobaric mass spectrometry MS quantitation of cysteine containing peptides lodolMT reagents showed efficient and specific labeling of peptide cysteine residues with reactivity similar to iodoacetamide Using anti TMT antibody we characterised the immunoaffinity enrichment of peptides labelled with iodoTMT reagents from complex protein cell lysates and for detection of S nitrosylated cysteines Introduction Thermo Scientific Tandem Mass Tag TMT Reagents enable concurrent identification and multiolexed quantitation of proteins in different samples
62. ip is sealed Test set up In this technical note we demonstrate the benefits of the revolutionary Clip Tip interlocking tip attachment technology by pipetting into a 96 well plate as an example Test pipettes and tips Thermo Scientific F1 ClipTip 12 channel pipette 30 300 ul with ClipTip 300 3001112 pipettes from Manufacturers and with manufacturers tips recommended for the particular pipette as well as generic tips The pipettes had friction based tip sealing mechanisms Test method Tip attachment was achieved with constant forces using a machine to press the tested pipette downward against the tip rack box with a lever The lever was equipped with a weight which directed a constant downward force to the test pipette This procedure simulated the tip attachment procedure with a known force The used attachment weight forces were 3 8kg 4 8kg and 6 3kg 97 3N 47 1N and 61 8N respectively After tio attachment 100 ul of green dye solution was dispensed into 96 well microplates using the reverse pipetting technique After each dispensing the tips were touched against the wall of the wells to wipe off possible droplets on the outside ofthe tip It was recorded if tips fell off After filling two microplates 100 ul was aspirated and the amount of liquid was measured gravimetrically Precision values CV96 were calculated for each attachment force used
63. ired to protect samples Heat discharge is a normal function of compressor driven units The heat generated can cause significant strain on HVAC systems HVAC is taxed to the limit which causes additional cost to run the entire building system Whatis the best time or application to consider putting water cooled condenser into a building Newconstruction projects Design Phase e Renovation e Locations that have existing recirculating water systems or chilled water source e Multiple freezers or freezer farms Advantages to water cooling condensers e Overall energy savings potential 5096 heat reduction to the laboratory or surroundings e Reduced impact on the HVAC system e comfortable work environment in the lab yields more productive work Heat reduction in the laboratory reduces potential risk to heat sensitive products or chemicals How do chilled condensers function The air cooled condenser looks like a small radiator like in your car The hot gas from the high stage compressor is circulated through the air cooled condenser where a fan blows across it to remove the heat and blow it into the room in air cooled condenser A water cooled condenser goes in place of the air cooled condenser and instead of a fan blowing across it coils of water is circulated around it which absorbs most of REFRIGERANT IN REFRIGERANT OUT WATER IN Figure 1 Graphical example of a water cooled condenser the
64. ith the biotin technique Free Radic Biol Med 2009 46 2 119 126 S P et al Quantitative analysis of complex protein mixtures using isotope coded affinity tags Nat Biotech 1999 17 994 999 4 Murray et al Identification and quantification of S nitrosylation by cysteine reactive tandem mass tag switch assay Mol Cell Proteomics 2012 11 2 111 013441 Acknowledgments The authors would like to thank Dr Jennifer Van Eyk and Dr Christopher Murray Johns Hopkins for sharing preliminary data on S nitrosylation switch assay protein labeling conditions Bio Innovation Issue Figure 4 Termer quand ation ido T ple r REA quarditati n ration d mira pi TRAIT S200 8 8 Results We have developed and used an iodoacetyl TMT reagent iodo TMT to irreversibly label sulfhydryls of cysteine containing peptides for multiplex quantitation by LC MS Figure 1 Compared to the reagent workflow the iodo TMT reagent workflow is simpler since reducing agents are not removed from protein samples before labelling Figure 2 The iodo T MT reagents showed efficient and specific labeling of peptide cysteine residues with reactivity similar to iodoacetamide Figure amp lodo T MT reagents were also used for sixplex isobaric quantitation of cysteine containing
65. itivity accuracy E E independently controlled nozzles to dispense and capability of resolving post translational modifications reagents into any well on a microplate The PTMs Many protein biomarkers in biological samples 1 compact design of the microfluidic chip has are present in low concentrations picograms mL and it is enabled the Mantis and Tempest to have a necessary to concentrate and enrich them for downstream small footprint to save lab space mass spectrometric analysis Immuno enrichment is commonly employed however most conventional methods are laborious and not able to deliver reproducible enrichment of biomarkers expressed in such low concentrations MSIA tips M a g J et provide a simple and effective way to enrich and concentrate Extraction Kits MagJET nucleic acid purification kits utilise target proteins down to femtomole level magnetic bead based technology allowing for highly efficient nucleic acid isolation from a variety of samples at any throughput MagJET kits provide high purity DNA and RNA ready to use in routine and demanding downstream applications The proprietary high capacity paramagnetic particles are optimised to isolate nucleic acids with superior purity and yields compared to other kits on the market erior Results Tandem Mass Tags TMT Thermo Scientific Tandem Mass Tag Kits and Reagents enable a rapid and
66. l of organic impurities in the water at various intervals accomplish this the conductivity C1 of the product water is measured and the value stored in the water system s processor During recirculation the water is then sent through the system s UV bulb where it is irradiated with UV light This oxidises any organics present in the product water The oxidation of the organics creates ions which are then measured by a downstream conductivity cell C2 The amount of extra ions in the water is directly proportionate to the amount of organics in the water if the UV bulb is working properly The difference between the conductivity cells is calculated and a TOC value is displayed The accuracy of the TOC measurement depends on how well the UV bulb irradiates the water If the bulb is not fully illuminated the total amount of organics in the water will not be oxidised resulting in a false reading To protect against this Thermo Scientific engineers created a photo electrode that directly monitors the UV lamp and ensures that it is working properly If there is a problem the system is designed to display an error If your application demands on extremely low levels of organics UV intensity monitoring can help ensure that your TOC measurements are accurate Thermo Scientific Luminaris Color Mis an advanced line of high performance qPCR master mixes with built in multicolour system for visual control over pipetti
67. lymerases are their extremely low error rate It is 50 fold lower than that of Taq polymerase Two different buffers are provided with Phusion DNA Polymerases Phusion HF Buffer error rate 4 4 x 10 and Phusion GC Buffer error rate 9 5 x 10 HF Buffer should be used as the default buffer for high fidelity amplification However GC Buffer can improve the performance some difficult or long templates Due to their high accuracy Phusion DNA Polymerases can reliably be used for studying mitochondrial DNA point mutations In conclusion Phusion DNA Polymerases perform accurate and fast amplification of mitochondrial DNA The conditions for Phusion High Fidelity DNA Polymerase and Phusion Hot Start DNA Polymerase were as follows 50 ng template 0 5 uM primers e200 uM dNTPs e 1 x GC buffer U enzyme Total volume 50 uL The cycling conditions for Phusion High Fidelity DNA Polymerases were as follows Acknowledgements Temperature Time Number of cycles We thank DocentAnu Wartiovaara Research 98 305 1 programme of Neurosciences 98 105 30 cycles University of Helsinki Finland for 72 8min 155 the primer sequences 72 C 4 C 10 min hold 1 Reference Tengan C H and Moraes C T 1996 Detection and analysis of mitochondrial DNA deletions by whole genome PCR Biochem Mol Med 58 130 134 www thermofisher co nz bio innovation www thermofisher com au bio
68. ment for a given application In addition frequent handling of delicate optics components presents a costly risk for instrument failure As a result many industrial laboratories choose to outsource complex analyses These limitations inevitably slow the laboratory s ability to respond to urgent business needs The Thermo Scientific M Nicolet 5 M50 FT IR spectrometer alleviates many of these productivity concerns by automating setup of the FT IR system for multi spectral range experiments 220 000 cm to 80 cm and for integrating techniques like FI Raman near IR and mid far IR attenuated total reflectance ATR into a single workflow Intelligent design behind the Nicolet i850 spectrometer permits unattended risk free operation increasing lab efficiency sample throughput and operational consistency between users This capability is delivered in an economical compact system 63 cm of linear bench space enabling any laboratory to employ multiple techniques for their analysis Flexibility and Value added Activities Working labs need analytical flexibility to respond to a variety of situations where answers are critical for decision making Examples include deformulating mixtures to build a case for patent infringement identifying counterfeit materials for product safety alerts analysing forensic samples for criminal investigations performing failure analysis to minimise production run delays assessing pro
69. methods References 1 Nelson R W Krone J R Bieber A L Williams P Mass spectrometric immunoassay Chem 1995 67 1153 1158 2 Niederkofler E E Kiernan O Rear J et al Detection ofEndogenous B Type Natriuretic Peptide at Very Low Concentrations in Patients With Heart Failure Cir Heart fail 2008 4 258 264 3 Lopez Rezai Sarracino D A et al Selected Reaction Monitoring Mass Spectrometric Immunoassay Responsive to Parathyroid Hormone and Related Variants Clin Chem 2010 56 281 290 2 4 Monzavi R Cohen P IGFs IGFBPs role in health and disease Best Pract Res Clin Endocrinol Metab 2002 16 433 447 5 Hankinson S E Willett W C Colditz G A etal Circulating concentrations of insulin like growth factor I and risk of breast cancer Lancet 1998 351 1393 1396 6 Wolk A Mantzoros S Andersson S O et al Insulin like growth factor 1 and prostate cancer risk population based case control study J Natl Cancer Inst 1998 90 911 915 A AMC of whole human mitochondrial DNA Here we compare the performance of five different polymerases when amplifying whole human mitochondrial DNA Thermo Scientific Phusion High Fidelity Polymerases were compared to three enzyme mixes recommended for amplification of long templ
70. mixes from two other vendors were compared by amplifying the whole human mitochondrial DNA Total DNA isolated from human blood was used as atemplate The amplified product was 16 5 kb Polymerase Amount Cycling time 1 Thermo Scientific Phusion High Fidelity DNA Polymerase 1U 4h48 min 2 Phusion Hot Start High Fidelity DNA Polymerase 1U 4h48 min 3 Enzyme mixture from supplier R 2 6U 7140 min 4 Enzyme mixture from supplier T 2 51 7 h 49 min 5 Enzyme mixture from supplier T hot start version 2 5U 7149 min he primers used for the amplification anneal to mtDNA at the following positions forward 10 40 reverse 16 494 16 463 All reactions were conducted using conditions recommended by the manufacturers The amounts of enzymes in units and total cycling times are shown in Figure 1 The reactions were set up on ice and run on a thermal cycler Conclusions disorders such as Parkinson s disease and Alzheimer s disease The performance of five different polymerases was compared when amplifying whole human mitochondrial DNA Phusion DNA Polymerases completed the entire PCR reaction in less than 5 hours while the other polymerases required almost 8 hours Compared to the three other polymerases tested which are recommended by the manufacturers for amplification of long genomic targets Phusion DNA Polymerases provided high yields with lower enzyme amounts An important advantage of Phusion DNA Po
71. n reproducibility reduces forces required to attach and eject tips and secures the best possible accuracy and precision In microplate applications demand for optimum pipetting performance and comfort is even greater due to higher number of repetitions and samples Compared to Manufacturer A and B the F1 ClipTip pipetting system demonstrates excellent precision and minimal variation between dispensed volumes of different channels Results show that pipetting performance of the F1 ClipTip system is not affected by the tip attachment force used tips remained attached and variation between channels was minimal In addition tip attachment did not require banging or rocking With friction based tip attachment systems the seal between the tip and the tip cone is accomplished by friction between the pipette and tip In this experiment impaired precision after dispensing and touching tips to the walls of amicroplate was observed This is likely due to loosening of the tips which affects the seal The sealing may be tight enough to avoid visible leakage but not tight enough to give optimal pipetting results In the worst case the tips fell off in the middle of pipetting In a real research application this would mean repeating the experiment Loss of samples and or reagent as well as time wasted have a large impact on daily research and can especially be seen with microplate applications Dropped tips may also present a i
72. n the porous monolithic solid supports First antibody is loaded onto the Protein A G surface The loaded antibody is then used to immuno capture and enrich its A Universal Mass Spectrometric Immunoassay MSIA Model system based on human insulin like growth factor 1 IGF 1 Sample dilution buffer 5 0 3 SDS Elution buffer 3396 acetonitrile O 496 trifluoroacetic acid Reduction buffer 10mM DTT in 30 isoproponal O 1M ammonium bicarbonate pH 8 0 Alkylation reagent 0 5M lodoacetamide O 1M ammonium bicarbonate pH 8 5 Thermo Scientific TSQ Vantage M Triple Stage Quadrupole Mass Spectrometer e Thermo Scientific Hypersil GOLD M C18 column 50 mm x 2 1 mm 1 9 um particle size Methods Samples An 8 point IGF1 calibration curve was prepared by serial dilutions of recombinant human IGF1 into standards targeted analtefrombiologial POMarker 27 dilution buffer concentration range 1 1500 ng mL IGF samples After rinsing retained Replicate samples n 12 from a single EDTA plasma donor A Materials and IGF1 calibration curve samples were prepared by diluting standard tryptic digestion and LC MS MS protocols Figure 2 An 8 point IGF1 from standard samples consisting of 1 5 10 25 100 500 1000 and 1500 ng mL of IGF1 and 500 ng mL of the internal standard LR3 IGF1 MS area ratios of IGF1 LR3 I
73. ng processes 9 T in your qPCR world The Luminaris qPCR Master Mixes are specially formulated to produce the most consistent and reproducible qPCR data using probe or SYBR Green chemistry across all real time platforms The Master Mixes contain Thermo ocientific Hot Start DNA polymerase in an optimised reaction buffer for increased reaction efficiency specificity and sensitivity Uracil DNA Glycosylase UDG included in the Master Mixes degrades the uracil containing PCR products carried over from previous experiments UDG for control over carry over contamination Due to the high sensitivity of qPCR even minute amounts of contaminating DNA can lead to false positive results The UDG enzyme included in Luminaris QPCR Master Mix degrades possible carryover amplicons from previous qPCR runs This ensures amplification of only specific products from the sample and not from the environment DNA spike compromises qPCR results 1ng 10 pg NTC E B B P H NB BH NEM E i HN E Gri UDG removes DNA spike prior GPCR id Li 15 La E ms Awe Start chemistry optimised buffer system for specificity sensitivity and reproducibility Luminaris Color qPCR Master Mixes are created to provide the highest specificity in GPCR The chemically modified Hot Start Tag DNA polymeras
74. ns z 2 and FIGURE 2 A representative example of the analysis is shown in Figure 3 The detailed structural information obtained from the MS MS data further validated the ability of the GlycanPac AXH 1 column to separate glycans based on charge size and polarity However coelution of different charge state glycans Figure 4 is common with other commercially available HILIC columns LC MS Analysis of Native Glycans Released from Proteins The GlycanPac AXH 1 column is well suited for high performance LC MS separation and analysis of native glycans from MAbs and other proteins Analysing unlabelled glycans not only eliminates the extra reaction step and cumbersome cleanup methods during labeling but also retains the original glycan profile without adding further ambiguity imposed by the labeling reaction Figure 5 shows the LC MS analysis of native N glycans from Bovine fetuin using the GlycaPac 1 column 1 9 The native glycans were separated based on charge size and polarity Using an ammonium formate acetonitrile gradient highly compatible with MS detection the separation enables excellent MS and MS MS fragmentation data for accurate confirmation of the glycan structure of each chromatographic peak Native glycan profiles are significantly Coe cee Dua FIGURE 4 SimGlycan software was used for glycan structural elucidation different from the profile of fluorescently
75. nswers needed for time sensitive decisions With a single user interaction the instrument can perform multiple measurements and analyses resulting in a final report even when unattended The Thermo Scientific OMNIC software provides a user friendly interface to set up applications quickly and generate spectra for definitive answers By adding powerful analytical tools like the Thermo Scientific OMNIC Specta software with a library of over 30 000 spectra and multi component searching or the TQ Analyst software for chemometrics a complete analytical workflow from sampling to results can often be achieved in less than 60 seconds This paper will demonstrate how the integration and automation of the Nicolet iS50 spectrometer leads to new levels of productivity while minimising risk to costly 1550 instrument becomes simpler as modules are added and as more manual steps are removed even when unattended FIR ATR Raman MIR ATR GC Measure Mjentify Gwantity Deformulaton Morphology Answer Figure 1 Nicolet i550 analysis work Automated Multi spectral Analysis Mid and Far IR ATR plus Near IR Most FT IR users understand the utility of the mid IR spectral range for qualitative and quantitative analyses Less well known the far IR region can provide new and unique information Simply put as the mass of atoms involved in vibrations increases the wavenumber decre
76. ore at one time This can be problematic pandemics e g the recent H1N1 swine flu outbreak are not easily predicted However since individual doses need to remain 5 8 cm away from walls doors drawers and cold air vents as these are most likely to have temperature extremes the footprint of the freezer needs to be chosen with care Bl gt Conclusion References 1 UNICEF Available at www unicef org immunization index coverage html 2 Welte M Vaccines ruined by poor refrigeration USA Today 2007 3 NHS National Patient Safety alerts www nrls npsa nhs uk alerts 4 CDC Notice to readers Guidelines for maintaining and managing the vaccine cold chain MMWR 2003 52 42 1023 1025 5 Thermo Scientific Laboratory Refrigerators and Freezers Guide for Vaccine Storage 6 UNICEF Supply Division annual report page Available at www unicef org supply index report html 7 Jeremijenko A Kelly H SibthorpeB et al Improving vaccine storage in general practice refrigerators BMJ 1996 312 1651 1652 8 Bell KN Hogue CJR Manning C etal Risk factors for improper vaccine storage and handling in private provider offices Pediatrics 2001 107 e100 9 Don t be guilty of these errors in vaccine storage and handling Immunization action coalition www immunize org The author Alex Esmon PhD Global Commercial Manager Laboratory Refrigerators and Freezers Laboratory E
77. ounting which gave similar results data not shown Figure 3 www thermofisher co nz bio innovation www thermofisher com au bio innovation FIGURE 4 iodoTMTsixplex reagent relative quantitation of BSA peptides Reduced BSA 100 ug was labelled with 10 mM iodoTMTsixplex reagent and combined in fixed ratios 126 127 128 129 130 131 10 3 1 1 3 10 amp 1 10 1 10 1 10 before protein digestion Graph of peptide relative quantitation for all quant FIGURE 5 Anti TMT enrichment of iodoTMT labelled peptides A Percent of iodoTMT labelled peptide modifications identified before and after enrichment B Comparison of unique proteins identified from unique peptides from A549 cell lysates before and after anti TMT antibody resin enrichment FIGURE 5 cysTMT and iodoTMT reagent labeling of S nitrosylated proteins A549 cell lysates A and BSA B were blocked with MMTS lanes 1 8 10 15 or untreated lanes 9 16 before 500 mM nitro glutathione NO treatment and TMT reagent labeling in the presence or absence of ascorbate and copper sulphate 504 Proteins were separated by SDS PAGE and analysed by anti TMT antibody Western blotting or Coomassie stain References 1 Thompson A et af Tandem mass tags a novel quantification strategy for comparative analysis of complex protein mixtures by MS MS Anal Chem 2003 75 1895 204 2 Forrester M T et al Detection of protein S nitroylsation w
78. ource Ken Vanoster Product Specialist Thermo Fisher Scientific Process to switch from house water source to chilled water source Check water pressure in both systems Pressure value should be x value Check the water flow rate If there is no information on water flow rate check the first stage discharge pressure Note the water pressure inlet water temperature and first stage discharge pressure Purge the chilled water line to remove air or particulates in the chilled water lines Check water pressure Must be at target pressure for chilled systems to protect the ultra low temperature freezers Switch the valve to chilled water Turn off house water source Monitor the temperature and water pressure on the chilled water line lt Udayanath Aich Ilze Birznieks Julian Saba Xiaodong Liu Rosa Viner Zhiqi Hao 2 Gurmil 5 Srinivasa Rao Andreas Huhmer Yury Agroskin and Chris Pohl Thermo Fisher Scientific Sunnyvale CA USA Thermo Fisher Scientifi c San Jose CA USA column technology tor trae LC MS analysis of labelled amp Native N CGlycans Heleased from Proteins amp Antibodies Methods Sample Preparation Helease native glycans from glycoproteins with PNGase F enzyme Conjugate the released glycans with 2 amino benzamide 2AB label group using the reported procedure of Bigge et Here 2 A1 P N GKSB 31 1 2 AB A2 P N GKSB 312 and 2 AB A3 P N
79. place on the luminometer It also shows how luciferase reporter gene assays provide excellent performance using low assay volumes with normal 384 well and shallow well 384 plates New lools tor Reporter Assays Luciferase genes are commonly used as reporter genes in gene regulation studies Reporter gene assays are used both in basic research for studying transcriptional regulation and cell signalling and in drug discovery for screening and validating drug targets The light output from luciferase reactions can be detected with luminometers that are suitable for reporter gene assays Thermo Fisher Scientific has developed a family of luciferase activity assays that use novel luciferase genes from Cypridina and Gaussia combined with the Red Firefly luciferase gene and the green shifted Aeni a gene The assay family provides both flash and glow type assays which differ in the stability of luminescence signals and in sensitivity Some of the assays utilise luciferases that are secreted into the culture media This feature allows live monitoring of the reporter activity during cell growth without a need for cell lysis In addition the assay family includes unique dual luciferase assays for multiplexing purposes he dual assays include two luciferases one for measuring the experimental luciferase activity and the other for measuring control activity for normalisation Bio Innovation Issue 7 gt Assay Types 1 Glow type lu
80. plications to ensure that the required centrifugation force does not exceed the manufacturer s specified g force rating gt B Fit of the conical tube in the centrifuge rotor It is important to note that the centrifuge tube performance largely depends on how well the tube fits in the rotor or rotor adapter For optimal tube performance it is imperative that there is contact between the tube and the rotor adapter so that centrifugal force can be distributed to the rotor rather than the tube Forces exerted directly on the conical tube may cause stress lines bulging or cracking For example when using a swing out bucket rotor in a centrifuge unit the direction of the g force centres on the conical bottom of the tube locking the tube in the centre position of the rotor In some cases this does not allow the lower portion of the tube to make contact with the rotor adapter The centrifugal forces on the side wall of the conical tube cannot be transferred to the rotor and may cause damage to the lower portion of the tube Figure 1 When using a fixed angle rotor in a centrifuge unit the side of the tube makes contact with the rotor adapter and most of the centrifugal force exerted on the tube can be transferred to the rotor Figure 2 Consequently the conical tube in a fixed angle rotor can be texture colour and shape of the plastic Chemical resistance is influenced by temperature duration and frequency of
81. poulos thermofisher com Art amp Design Andrew Dennis andrew dennis thermofisher com am delighted to welcome you to our 7th edition of the Bio Innovation series an educational publication focused on new and emerging trends for every life scientist in Australia and New Zealand This edition of Bio Innovation focuses on the most recent technology platforms and products released to market by Thermo Fisher Scientific Thermo Fisher Scientific recognises that the local landscape for scientists is becoming more challenging more competitive from a funding perspective By investing in our life science community with publications such as this we hope to assist you to increase efficiency and give you the freedom to focus on your research Our goal is to help you succeed However you measure success we develop the technology platforms provide the application resources and the programs to help you achieve it Our organisation is built on over a century of experience both locally and globally We are over 750 people in Australia and New Zealand working together to deliver innovation and create connections across our portfolio to accelerate customer results and serve science in ways no one else can One of the great things about our organisation is our mission We enable our customers to make the world healthier cleaner and safer see this in practice often travel to our customers laboratories and see our technology platforms from
82. quipment Division Thermo Fisher Scientific With organisations investing much time effort and money to screen develop and produce potentially life saving vaccines it is detrimental in terms of time and cost efficiency if these vaccines lose effectiveness through incorrect storage Maintaining constant conditions for vaccine storage is key to improving viability and protecting a population from health risks Selecting the most efficient clinical cold storage equipment is therefore crucial for ensuring sample protection The latest available refrigeration equipment enables much easier and more reliable monitoring and maintenance of ideal temperature conditions for vaccine storage reducing the occurrence of temperature fluctuations to a minimal level These in built technological advances in storage units in combination with a degree of education on quality control measures can help to ensure that vaccines remain viable after storage eliminating the need for costly losses and the implementation of revaccination programmes www thermofisher co nz bio innovation www thermofisher com au bio innovation Heija Riitta Harinen Jorma Lampinen and Arto Per l Thermo Fisher Scientific Vantaa Finland Janaki Narahari and Douglas Hughes Thermo Fisher Scientific Pierce Protein Research Rockford IL USA The focus of this application note is on introducing different luciferase reporter assay types and the requirements they
83. r collection of the unbound sample the resin was washed 4X with 4 M Urea TBS 4X with TBS and 4X with water Peptides were eluted 3X with 50 acetonitrile O 496 TFA frozen and then dried under vacuum before LC MS MS analysis Selective labeling of S nitrosylated proteins Proteins and cell lysates were solubilised at 2 mg mL in modified HENS buffer 100 mM HEPES 8 0 1 mM EDTA 0 1 Data Analysis MS spectra were searched using Thermo Scientific Proteome Discoverer software v1 3 o m Proteirvpeptide with modified cysteine Figure 2 i cuni Ogest tute Ennch for TMT tagotd lor preter pepb e using anti TMT resin mM neocuporine 1 SDS Free sulfhydryls were blocked with methyl methane thiosufate MMTS for 20 min at room temperature and desalted to remove excess blocking reagent S nitrosylated cysteine sulfhydryls were selectively labelled using O 4mMiodoTMT reagent in the presence of 20 mM sodium ascorbate Excess iodoTMT reagent was removed by acetone precipitation of samples at 20 C for 4 20 hrs Unlabelled cysteines were reduced and alkylated with 20 mM iodoacetamide Proteins were enzymatically digested at 37 C for 4 hrs and desalted before LC MS MS analysis or enrichment LC MS MSAnalysis A nanoflow high pressure liquid chromatography system with a Thermo Scientific PepMap C18 column 75 um ID x 20 cm was used to separate peptides using a 5 40 grad
84. raded multi spectral range sys 1 canacquire spectr R modules a at the touch gi i initiate novel ATR Ramar techniques without man ial S FT IR bench to a W automated ging system compo on husion High Fidelity DNA Polymerases r Incorporating a unique dSDNA binding domain Phusion DNA Polymerase amplifies DNA with accuracy and speed unattainable with standard PCR enzymes even on the most difficult templates Phusion has a High fidelity error rate 4 4 x 107 in Phusion HF Buffer the speed of the polymerase allows short extension times 15 30 s kb the robust reaction means minimal optimisation needed and it produces increased product yields Thermo Scientific Pierce Luciferase Reporter Assay Thermo Fisher Scientific has developed a family of luciferase activity assays that use novel luciferase genes from Cypridina and Gaussia combined with the Red Firefly luciferase gene and the green shifted Renilla gene The assay family provides both flash and glow type assays which differ in the stability of luminescence signals and in sensitivity Some of the assays utilise luciferases that are secreted into the culture media This feature allows live monitoring of the reporter activity during cell growth without a need for cell lysis In addition the assay family includes unique dual luciferase assays for multiplexing purposes The dual assays include two lucife
85. rases one for measuring the experimental luciferase activity and the other for measuring control activity for normalisation Conical Centrifuge Tubes Premium high quality conical tubes that are environmentally friendly offer the highest cleanliness with a recyclable plastic rack and allow for increased traceability with the largest writing area on the market The Thermo Scientific Nunc tubes are the highest speed rated tubes on the market with our 15ml tubes rated to 10 500xg and the 50ml tubes rated to 17 000xg Thermo Scientific Nunc Labware Products are made from high purity resins and molded using our state of the art processes Plastic labware is a safer alternative to glass without sacrificing accuracy Large Scale Hui Li Department of Pharmacology and Molecular Toxicology University of Massachusetts Medical Center Worcester MA Preoaration Many different protocols can produce plasmid in large quantities that are sufficiently pure for general cloning enzyme digestion sequencing cellular transfection in vitro transcription translation protein expression and other purposes For instance DNA can be isolated in cesium chloride gradients but this method requires an ultracentrifuge and utilises ethidium bromide which must be handled with care Alternatively gravity or spin column kits effectively purify plasmid DNA in superspeed and microcentrifuges This brief highlights Thermo Scientific
86. ration continuous attention is needed to swap the beamsplitters at the right moments This seemingly hidden improvement allows unattended operation permitting productivity through automation Figure 2 shows just the mid and far IR spectra collected from acetylferrocene analysed using an OMNIC macro controlled workflow The additional information from the far IR spectra is clear the low end triplet verifies that the iron is sandwiched but the entire process required seven minutes including collection of the mid and far IR backgrounds Automation also reduced the total hands on time of the user pressing buttons loading sample to 20 seconds Multiple Techniques and Multi range Analysis _ Enhanced Flexibility The Nicolet 1650 spectrometer can be configured with FT Raman and wide range diamond ATR Switching between these experiments raises concerns of instrument recovery time purge exposure handling of optics and potential confusion or user error The experiments are often seen as independent activities for these reasons The spectrometer with 1550 ABX simplifies this apparently complex situation to one step initiation of a macro The Nicolet 1650 instrument shown in Figure is configured with the 1650 1550 Raman 1550 ATR and the 1550 ABX modules shows how easy sample loading and analysis can be done For operating one module at a time the user need only pres
87. ration i Scientific Selecting Centrifuge RNAzol Ameren Marginal may be satisfactory for use in Laboratory April 2009 Thermo a centrifuge depending on length of exposure Trypsin 5 Scientific NALGENE Centrifuge 500 and speed Testing under operating conditions is Toluene pure U Complete Guide to Centrifuge SUggested before actual run wem Ware 2001 U Unsatisfactory not recommended i aa ww The Thermo Scientific mYECL Imager offers a new easier and more efficient option to acquire Western blot and proteir nucleic acid gel data Easy set up with no engineering support necessary Touch screen controls with an intuitive workflow interface Open data formats for sharing and publishing simplicity Coupled with the Thermo Scientific mylmageAnalysis Software you can analyze your captured images quickly and accurately The MYECL Imager delivers efficiency and savings making your data and your lab happy in real time image p at one touch to your image no extra parameters to set one touch to quickly and easily share and export data Exposure Time L from your laptop 108 158 308 1m zm 5m one step to stronger signal intensity with our Western blotting reagents de 2 per lt o scan to an informative video on our imager 2012 Thermo Fist
88. reviously described cysteine reactive workflows for labeling enrichment and quantitation of cysteine containing peptides and cysteine modifications such as S nitrosylation Conclusions lodoacetyl Tandem Mass Tags iodoTMT are novel reagents for specific irreversible labeling of cysteine residues pre or post digestion reagents can be used as either isotopic pairs or as an isobaric set for MS or MS MS based multiplexed quantitation An antibody to the TMT reagent reporter region allows specific detection capture and enrichment of iodo T MT labelled proteins and peptides reagents can be used for detection of cysteine modifications such as S nitrosylation A New Ajax Finechem Product Catalogue Ajax Finechem Part of Thermo Fisher Scientific Throughout the long history of Ajax Finechem quality and innovation has underpinned the brand Ajax products are prepared in an 150 accredited laboratory and analytical grades are tested to ensure these products meet the most stringent specifications Thermo Fisher Scientific supplied Ajax Finechem products combine these qualities with a commitment to listen and respond to customers needs or KEEP EAD OUT SAFETY DIRECTIONS Order your copy of the Ajax Finechem NOT SWA 2508 2 5 New Catalogue is Now Available Catalogue now Simply visit www thermofisher com au ajaxcatalogue Pass it on if you have
89. rformed using a single Protein A G MSIA Tip loaded with antibody per each sample following the protocols provided in the user manual total processing time 30 minutes After extraction IGF1 and L R3 IGF1 were digested and analysed by SRM described below Sample Elution and Trypsin Digestion Captured IGF1 and R3 IGF1 were co eluted from the Protein A G MSIA Tips by repeatedly mixing 20 cycles of aspirating and dispensing 30 UL volumes 50 uL of elution buffer within the wells of a 96 well plate using the MSIA Tips with captured IGF1 and LR3 IGF1 thus eluting IGF1 and LR3 IGF1 into the 50 uL elution buffer within each well Samples were lyophilised to dryness and then re suspended in 30 uL of reduction buffer and allowed to reduce for 30 minutes at 37 C Reduced samples were then alkylated by adding 2 4 uL alkylation reagent and incubating in the dark at room temperature for 3O minutes Reduced and alkylated samples were diluted with 92 5 uL of warm 50 C 0 1M NH4HCO SmM CaCl and then digested by adding 25 uL 4 mg L trypsin to each sample Samples were allowed to digest for 2 hours at 50 C and then stopped by adding 5 3 UL of acid solution 8 uL 100 formic acid and 2 3 uL 1mg mL glucagon Injection volumes of 155 uL of the digests were injected into the LC MS for SRM SRM Methods SRM methods were developed on a Thermo Scientific TSQ Vantage Triple Stage Quadrupole Mass Spectrometer with a Thermo Sc
90. s diversity for a wide range of assays from those requiring factorial dispensing like assay design and modalit of inhibition studies to applications like assay miniaturisation that use fixed volume dispensing Tempest Assay Variable Volume The Tempest is a flexible and compact liquid dispenser that Avg Std dev CV Assay CV s uses state of the art microfluidics to dispense any volume 0 390 0 003 0 818 Kinase 1 318 from any input into any well 0 388 0 003 0 896 Kinase P 1 122 0 390 0 003 0 739 Kinase A1 1 133 It uses positive displacement to dispense liquids with minimal 7 0388 000 0805 Kinase F 1206 waste It has a dispensing range of 200nl to no upper limit Tm and a very low non recoverable dead volume 40ul Typical EE 12 Am me 62 5 em Nase dead volumes are 400yl and can be reduced to 100yl 4 ee 0 390 0 004 0 950 Kinase A1 2 519 using tip dispensing lt is accurate precise fast and is aaa compatible with 96 384 1536 well micro plates E ANE NM MIT E 0 390 0 003 0 754 Kinase FL 1 885 0 390 0 003 0 714 Kinase R 3 109 Tempest accurac MM p y 0 389 0 003 0 801 5 2 109 We analysed the accuracy of the Tempest in two formats e 0 389 0 003 0 789 Kinase V 1 905 A fixed volume miniaturised assay comprising of 2ul amp substrate A variable volume assay that requires different volumes dud oe RE A of 8
91. s the associated Touch Point Seen more closely in Figure 4 Touch Points make one button operation effortless when switching between modules sampling stations and automating optics exchange Rather than thinking through the components needed light source beamsplitter optical path and detector to run an experiment the user simply presses the Touch Point to switch from an ATR to measurement waits until the instrument indicates that it is ready to begin This error free operation is done in 30 seconds The Nicolet 1550 analytical power in Figure 1 becomes clear when the four data collections mid IR and far IR ATR NIR and Raman are between the cyclopentadiene rings The NIR data is not shown performed one workflow Collecting spectra from each of these modules using conventional manual approach required about 50 minutes including sample loading optical changes time for equilibration and optimisation Nicolet 1550 of the Raman signal The analyst Process Step Typical minutes with Built in ATR minutes needed to be presen t throughou t Sample Preparation Grind Mix 10 None 0 the experiment to perform the Mid IR Background Collect BKG 0 5 Collect BKG 2nd 1 beamsplitter changes and collect Mid IR Collect Load Sample 2 Load Sample 1 various backgrounds for each Collect Spectrum Collect Spectrum sampling station At the end of the Change Optics
92. s to be pipetted directly onto the optical measurement surface After measurement the sample is wiped off the measurement surface with a lint free lab wipe marter ITechnolc rogess ClipTips Thermo Scientific ClipTip pipette tips provide security with a unique and innovative interlocking FREIE technology that ensures a complete seal on every channel with minimal tip attachment and ejection force Achieve newfound confidence knowing I that once attached your tips are locked firmly in place and will not loosen or fall off regardless of pressure The innovative three _ 41 _ interlocking clip design ensures the tip is held a 1 on the F1 ClipTip pipette until and Hi 1 until it is released i ZA MAI d ARS I I eo YU us EU Thermo Scientific Mass i 1 Spectrometric Immunoassay ANM MSIA Tips he Thermo Scientific MSIA Tips are the next generation LUI ig A immunoaffinity approach that simplify peptide protein a redefines liquid handling dd by enrichment for downstream quantification using mass E replacing traditional methods with a spectrometers Mass spectrometry has become an important d oma HL microfluidic chip This technology utilises tool for biomarker research because of its sens
93. ther concentrations are given in table 1 It can be seen from Fig 8 and 9 that for both Inhibitor A and Inhibitor B the potency of inhibition decreases with increase in ATP concentration a characteristic of Competitive Inhibition Figure 6 Figure 7 0 4 15 250 ATP 15um ATP 250 EC50 401 9 1953 50 38 15 258 6 s 15uM ATP 15uM ATP 0 2 250uM 250uM 0 1 Figure 8 Figure 9 300 200 100 0 0 5 10 15 20 S Km S Km Inhib A IC50 nM ATP uM Inhib IC50 nM 258 250 1953 151 125 1300 91 62 5 882 65 31 25 530 36 15 402 27 7 5 oor 2 3 75 258 10 1 75 119 Table 1 Cell dispensing We further evaluated whether operation of the diaphragm during dispensing had an effect on cells As shown in Fig 10 amp 11 we saw no morphological changes in cells dispensed with the Tempest when compared to manual dispense Low volume pipetting technology is key to our workflow and is of utmost importance Here we have highlighted the improved efficiency the Tempest provides to our workflow Other key features include an easy USB connectivity a user friendly software and automation capability ELISA PCR HTS DNA RNA research are some of the other applications that the Tempest can support Figure 1 Kinase A enzyme titration at 100uM ATP Figure 2 Kinetics for Kinase A Figure 3 Kinase A enzyme titration at 15uM ATP Figure 4 Kinetics for Kinase A TE
94. tinct wavelengths The signals can easily be separated using a luminometer equipped with high transmission filters optimised for the assays This kind of dual assay eliminates the need for signal quenching between the measurement of two luciferases Thus both luciferase signals can be detected simultaneously from a single sample with a single reagent addition As these dual assays are flash type assays the reagent dispensers installed in the luminometer act to ensure optimal performance and sensitivity gt Luminometers Luminescence light output from luciferase reporter gene assays can be quantified using for example Thermo Scientific Varioskan Flash or Luminoskan Ascent microplate luminometers Figure 1 These luminometers have excellent sensitivity in luminometry both in glow and flash type assays In addition the Varioskan Flash can measure luminescence spectra as well as other detection technologies Reagent dispensers amp Optical filters Dispensers are essential in flash type assays which require measurement soon after the addition of substrate and precise timing between dispensing and measurement from sample to sample and from day to day Dispensers installed in the luminometer make the reaction triggering easy fast and accurate not only for flash type assays but also for glow type 3 assays Both the Luminoskan Ascent and Varioskan Flash can be equipped with up to three reagent dispensers In addition
95. tion of rotors and adapters for the Sorvall LYNX 6000 superspeed centrifuge that will serve to accommodate the forces required by most commercial kits Thermo Scientific Rotor Capacity MaxSpeed MaxRCF place x mL rpm 9 Fiberlite F14 14x50cy Table 3 14x50 14 000 33 746 Fiberlite F14 14x50cy with adapter 14x15 14 000 33 746 24 15 21 500 63 049 A21 24x15C TH13 6x50 with adapter 75007322 6x50 13100 30 314 Conclusion This application brief de superspeed offerings plasmid DNA preparation accommodates small and larg and offers efficient versatile ligh Table 1 Solutions for DNA preparation Table 2 Rotors available for DNA preparation in the Sorvall LYNX 6000 superspeed centrifuge Table 3 High speed 15 mL and 50 mL conical tube rotors and adapters available for DNA preparation in the Sorvall LYNX 6000 superspeed centrifuge References 1 Noles S R Schwartz M W Fischer R A Martin R Schneider W Jagadeeswaran P Rao K J 2008 Traditional Methods for CsCI Isolation of Plasmid DNA by Ultracentrifugation Selecting Conical Tubes for Centrirugation Applications By Stephanie Carter Wenxiao Lu Marci Moore Joe Granchellt amp Cindy Neeley Laboratory centrifuges are common everyday instruments While centrifuge usage 15 widespread the selection of the proper centrifuge and rotor system along with the proper centrifuge tube cons
96. ts in a 96 well plate format provide excellent performance as well The performance of the Luminoskan Ascent with these luciferase reporter assays is also comparable to the Varioskan Flash results presented here 7 A Urn AIR 1 iy i Highly sensitive flash type assays can easily be performed with a microplate luminometer equipped with an automatic dispenser Both inter and intra assay variations can easily minimised using automatic luminometer dispensers which enable precise timing of the reaction start and signal collection Different luciferase reporter gene assays require different features and capabilities of the luminometer The benefits of gt Conclusions Pierce Luciferase assays provide an extremely wide dynamic range for measuring both low and high luciferase expression levels without concentrating or diluting samples The Varioskan Flash detects luciferase activities over a dynamic range of about eight orders of magnitude with the automatic gain adjustment feature of the instrument Using 384 well plates either normal or shallow well enables reducing both sample and reagent consumption without sacrificing assay performance Use of shallow well 384 plates enables performing the assays in a total volume of about 6 ul 1 ul sample 5 ul assay reagent i2 Kinetics of Gaussia flash assay 060 050 or ar ae a ee
97. umable can often be a challenging experience There are several factors to consider when selecting the correct conical tube for centrifugation These include the relative centrifugal force required the fit of the particular tube in the rotor sample volume and the compatibility of the sample to the tube material Understanding the requirements of a centrifugation application before selecting a conical tube can prevent sample leakage or loss allow for easy sample recovery and reduce the risk for potential damage to the centrifuge and rotor A RPM vs RCF All centrifuge tubes have a maximum speed rating determined by the manufacturer Vessels used at speeds higher than the recommended rating can fail resulting in sample loss and potential damage to the centrifuge and rotor Most protocols specify speed in either revolutions per minute RPM or relative centrifugal force RCF It is crucial to understand the difference between HPM and RCF The rotor revolves at the specified RPM and the force applied to the contents is dependent on the rotor s radius with larger radii applying greater force on the sample RCF represents the gravitational force being applied to the sample It determines the centrifugation outcome independent of rotor size RCF is measured in force x gravity or g force and is more relevant to the actual impact and outcome of the centrifugation than RPM Users should verify the required RCF by their specific ap
98. using tandem mass spectrometry Previously we described a cysteine reactive isobaric Tandem Mass Tag cysTMT reagent that utilised a dithiopyridine reactive group to selectively label cysteine sulfhydryls Although this labeling chemistry is highly specific and efficient it results in a reversible di sulfide linkage between the peptide and isobaric tag we report the development of an irreversible cysteine reactive TMT reagent containing an iodoacetyl reactive group iodo TMT Due to the irreversible labeling of the iodoTMT reagent it can be used for quantifying cysteine modifications such as S nitrosylation oxidation and disulfide bridges Sample Preparation Preparation of iodoTMT labelled proteins Proteins and or cell lysates were solubilised at 2 mg mL in 50 mM HEPES pH 8 0 0 196 SDS and reduced with 5 mM for 1 hour at 50 C Reduced proteins were labelled with 5 10 mM iodoTMT reagent 10 molar excess for 1 hr at 37 C protected from light Excess iodoT MT reagent was removed by acetone precipitation of samples at 20 C for 4 20 hrs Proteins were enzymatically digested at 37 C for 4 hrs and desalted before liquid chromatography LC MS MS analysis or enrichment Enrichment of iodoTMT labelled peptides Labelled peptides 25 100 ug were resuspended in TBS at 0 5 ug uL and incubated with an immobilised anti antibody resin 20 100 UL overnight with end over end shaking at 4 C Afte

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