Protein A280 - Thermo Scientific
Contents
1. SCIENTIFIC Part of Thermo Fisher Scientific The information in this publication is provided for reference only All information contained in this publication is believed to be correct and complete Thermo Fisher Scientific shall not be liable for errors contained herein nor for incidental or consequential damages in connection with the furnishing performance or use of this material All product specifications as well as the information contained in this publication are subject to change without notice This publication may contain or reference information and products protected by copyrights or patents and does not convey any license under our patent rights nor the rights of others We do not assume any liability arising out of any infringements of patents or other rights of third parties We make no warranty of any kind with regard to this material including but not limited to the implied warranties of merchantability and fitness for a particular purpose Customers are ultimately responsible for validation of their systems 2010 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries Copyrights in and to the technical support image are owned by a third party and licensed for limited use only to Thermo Fisher Scientific by iStockphoto No part of this publication may be stored in a retrieval system transmitted or reproduced in any way including but not l2. Tip Use this option when the molar extinction coefficient M cm and molecular weight are known Other protein 1 e Enter the mass extinction coefficient L gm cm for a 10 mg mL 1 solution of the respective reference protein Calculations Pathlength amp Concentration Thermo Scientific NanoDrop Spectrophotometers Protein A280 NanoDrop instrument pedestal measurements utilize pathlengths of 1 0 mm to 0 05 mm model dependent As the pathlength gets shorter the ability to measure higher concentrations without saturating the internal detector increases Although the upper detection limit of the internal spectrometer is 1 5 Absorbance units the NanoDrop sample retention technology allows for the use of shorter pathlengths thereby extending the absorbance range of the instrument The graphic below illustrates how utilizing pathlengths less than the standard 10 mm cuvette pathlength enables higher concentrations of samples to be measured without making sample dilutions 10 mm pathlength 1 mm pathlength 0 2 mm pathlength max concentration max concentration max concentration 2 mg mL BSA 20 mg mL BSA 100 mg mL BSA 10 mm pathlength Equivalent to max Abs value Equivalent to max Abs value max Abs value 1 5 of 15 when normalized to a of 75 when normalized to a 10 mm pathlength 10 mm pathlength The table below lists the BSA detection limits specific to each model Model Absorbanc
3. A280 Troubleshooting Reproducibility Concentrations not within Ensure samples fall within the linear detection range of Expected Range the instrument Tip Refer to the table of model specific detection limits on page 17 for guidance e Ensure sample solution is homogeneous by gentle vortexing as appropriate e Confirm that the reference blank solution and sample solvent are the same material e Clean and recondition the pedestal surfaces prior to the start of the measurement session e Ensure appropriate sample type is selected as concentration calculations utilize constants specific to each sample type Instrument Related Issues Column Breakage e Ensure pedestal surfaces are properly conditioned Tip When a pedestal becomes unconditioned sample droplets applied to the bottom pedestal will flatten out and cover the entire pedestal surface rather than bead up Refer to the Reconditioning instructions under the Best Practices section on page 6 e Ensure sufficient volume is loaded onto the pedestal e Use a larger volume 1 5 2 uL for each measurement e Use a calibrated small volume pipettor to deliver the sample to the pedestal e Ensure instrument is not located near a vent or other source of air flow e Ensure measurements are made immediately after pipetting samples onto the pedestal as delays may compromise accuracy e f an error message indicating possible column breakage is di
4. definition of system would include the instrument protocols being used techniques employed by the user and the solution utilized as the control e A2 ug ml Bovine Serum Albumin solution is a routine laboratory control used to monitor the reproducibility and values obtained from day to day use e Controls are valid to use as long as the instrument is calibrated and the control product itself is within the expected concentration range stated in the manufacturer s specifications Tip Ensure all controls are stored as recommend by the manufacturer Do not use controls past the stated expiration date o D gt 9 Thermo Scientific NanoDrop Spectrophotometers Protein A280 Troubleshooting Common Reagents 10mm Absorbance 10mm Absorbance When troubleshooting sample measurements it is important to utilize the sample spectrum as a primary guide 15 4 10mm Absorbance 10 0 220 230 240 250 260 270 280 290 300 310 320 330 340 350 Wavelength nm Typical Protein Spectrum at 280 nm BSA in PBS Below are several examples of reagents commonly used with proteins with absorbance in the 220 350 nm range 20 a 0 54 0 10mm Absorbance 2 220 230 240 250 260 270 280 290 300 310 320 330 340 350 115 100 754 50 254 0 Wavelength nm RIPA Buffer 10mm Absorbance 10 ___ __ _ __ oe 220
5. to make measurements are the same across all wavelengths Therefore when pathlengths are verified at one wavelength the verification is valid for the entire measured spectrum Calibration Check Fluid e CF 1 is a standard manufactured exclusively for use with NanoDrop Spectrophotometers and is available from Thermo Fisher Scientific and its distributors e The CF 8 Calibration Kit used for the NanoDrop 8000 calibration check procedure includes 2 CF 1 vials as well as 8 well PCR strip tubes Tip It is good practice to check the instrument s performance every six months with a new vial of NanoDrop Calibration Check Fluid Calibration Standard vs Control Thermo Scientific NanoDrop Spectrophotometers Protein A280 A Standard is generally accepted as a solution of a known concentration that is used to calibrate or certify that an instrument is working within acceptable pre defined guidelines e The NanoDrop CF 1 Calibration Check Fluid is the only acceptable standard for use with the NanoDrop instrument Calibration Check diagnostic available within the operating software e The term Standard also refers to protein solutions of a known concentration used to define a standard curve These standards are not appropriate to assess NanoDrop instrument performance A Control is a solution that produces an expected result within a specific range if the system is working as expected The
6. 0 Best Practices Angle the instrument for optimal pipetting Li ede Right handed orientation for the NanoDrop 2000 2000c and close up view of the NanoDrop 8000 4 saonoelg seg Cuvette Orientation Use etched arrow as light path guide when inserting quartz or masked plastic cuvette NanoDrop 2000c only Tip Locate instrument away from air currents and exhaust fans Thermo Scientific NanoDrop Spectrophotometers Protein A280 Use adequate sample volume to ensure good column formation Tip Always use 2 uL samples when measuring protein samples to ensure proper column formation Surfactants and other components routinely used in protein buffers may reduce the surface tension properties of the liquid Use calibrated pipettor with well fitting tips It is best to use a precision pipettor 0 2 uL with low retention precision tips to ensure that a sufficient sample 2 uL is delivered for optimal column formation Tip To avoid evaporation errors it is essential to use an eight channel pipettor to simultaneously load samples when using two or more pedestal positions on the NanoDrop 8000 Best Practices Volume Requirement Sampling Technique Pipettor Selection Sample Aliquots Always use fresh tips and fresh aliquots for every measurement Tip Repeated measurements on the same sample aliquot will result in evaporation yielding increasing concentrations and or column breakage Sample
7. 230 240 250 260 270 280 290 300 310 320 330 340 350 Wavelength nm 4M DIT 300 200 100 0 220 230 240 250 260 270 280 290 300 310 320 330 340 350 Wavelength nm Triton X 100 80 70 60 4 50 4 40 30 4 204 104 0 10 4 220 230 240 250 260 270 280 290 300 310 320 330 340 350 Wavelength nm 7M Urea and 2M Thiourea Thermo Scientific NanoDrop Spectrophotometers Protein A280 Troubleshooting Unusual Spectra Negative values associated with some spectra indicate that either the F pedestals were very dirty when the blank measurement was made or that a 3 sample was used to make a blank or reblank measurement lt 4 Suggestion PTICE CTE CET Clean pedestal and measure new blank Wavelength nm f2 D a amp 8 A ragged appearance throughout a spectrum may indicate a bad blank 3 Suggestion o 10 Clean pedestal and measure new blank 0 00 0 10 220 230 240 250 260 270 280 290 300 310 320 330 340 350 Wavelength nm A jagged appearance throughout a spectrum may indicate a broken column Suggestions Clean and recondition both measurement surfaces then measure new blank Absorbance Increase sample volume to ensure proper column formation 4 220 230 240 250 260 270 280 290 300 310 320 330 340 350 Wavelength nm A jagged appearance at t
8. 4 5 mg mL BSA Q What are the protein detection limits A The detection limits vary depending on the model and the protein type being measured The table below presents the limits by model for Bovine Serum Albumin with a mass extinction coefficient of 6 7 at 280 nm for a 1 10 mg mL solution Model Detection Limits for BSA NanoDrop 2000 0 1 mg ml to 400 mg ml NanoDrop 2000c 0 010 mg mL to 400 mg ml NanoDrop 8000 0 1 mg ml to 100 mg ml NanoDrop 1000 0 1 mg ml to 100 mg ml Denotes lower detection limit when using 10 mm path cuvette FAQs Thermo Scientific NanoDrop Spectrophotometers Protein A280 Q What if my samples are less concentrated than the above indicated lower detection limits A Fluorescent dyes can be used in conjunction with the NanoDrop 3300 Fluorospectrometer to detect protein samples less concentrated than the limits indicated in the table on page 26 Refer to the NanoDrop 3300 Fluorospectrometer user guide found under the Support tab on www nanodrop com Q What sort of accuracy should expect with the NanoDrop 2000 2000c A Typically within 2 at the 1 mm pathlength Q What sort of reproducibility should expect with the NanoDrop 2000 2000c A Typically 0 1 mg mL for samples lt 10 mg mL and 2 for samples gt 10 mg mL BSA samples Q Is simply wiping the pedestal surface enough to prevent carryover A Yes The highly polished quartz and stainless steel surfaces of the sample ret
9. Homogeneity Highly concentrated protein samples require careful attention to ensure homogeneity before sampling Tip Non reproducible results observed when making small volume measurements are a good indicator that the sample is not fully in solution or is not homogenous Tip It may be necessary to lightly vortex samples prior to sampling to ensure homogeneity Sample Preparation Ensure sample isolation procedure is optimized and sample is purified prior to measurement Any molecule with absorbance at 280 nm will contribute to the total absorbance used to calculate sample concentrations Triton X 100 and NP 9 are two examples of components found in common buffers that may contribute to the total absorbance values at 280 nm Measurements Blank Measurements Thermo Scientific NanoDrop Spectrophotometers Protein A280 Pre assessment of buffer compatibility with direct A280 measurements e Many buffers commonly used with protein samples contain components with significant absorbance in the UV region Proteins in these types of buffers may not be suitable for direct quantitation using the A280 method e Proteins suspended in RIPA buffers may not be suitable for measurement using the A280 application It is recommended that a colorimetric assay such as Pierce 660 nm be used for proteins suspended in RIPA buffers Refer to the reagent manufacturer for more information regarding buffer compatibility Follow the steps below
10. Tip Although it is not necessary to blank between each sample it is recommended that a new blank be taken every 30 minutes when measuring many samples D A a Measurements Sample Measurements Thermo Scientific NanoDrop Spectrophotometers Protein A280 1 After the blank measurement is complete enter the sample name in the Sample ID box and choose the appropriate sample type as described below Sample Type Option Extinction Coefficient BSA 6 67 IgG 13 7 Lysozyme 24 6 1 Abs 1 mg mL Default general reference setting E1 User entered mass extinction coefficient E and MW User entered molar extinction coefficient and molecular weight Refer to Sample Types Options in the Calculations section on pages 15 and 16 for more information 2 Pipette an aliquot of the protein sample onto the lower measurement pedestal and lower the sampling arm Click Measure Tip If measuring more than one sample at a time on the NanoDrop 8000 it is important to use a multi channel pipettor to deliver the sample aliquots 3 After the measurement is complete use a dry lint free lab wipe to remove the sample from both the top and bottom measurement surfaces 4 Change pipette tips for the next measurement Tip If measuring multiple replicates of the same sample it is important to use a fresh aliquot for every measurement to ensure accurate results 5 Review spectral image to assess
11. contact Technical Support Outside of the US and Canada please contact your local NanoDrop products distributor Troubleshooting Connection Errors Signal Errors Thermo Scientific NanoDrop Spectrophotometers Protein A280 If your instrument operates properly most of the time but connection errors appear intermittently the instrument may not be receiving power or recognizing the USB connection e Ensure that the USB and power cables are plugged into the back of the instrument and that the instrument is receiving power e Many instrument issues can be addressed by a simple power restart 1 Exit the software 2 Disconnect the instrument power cord and USB cable 3 Reconnect the instrument power cord first then the USB cable 4 Restart the software If the error persists contact Technical Support Outside of the US and Canada please contact your local NanoDrop products distributor Some error messages are triggered when little to no light reaches the detector during initialization or a measurement e Refer to the cleaning directions under the Best Practices section on page 6 e Run the Intensity Check diagnostic Refer to the model specific user guide for additional information 25 26 FAQs Thermo Scientific NanoDrop Spectrophotometers Protein A280 Q Can quantify proteins using methods other than the A280 application on NanoDrop spectrophotometers A Yes Other option
12. e Detection Limits Typical Reproducibility Limits for BSA minimum 96 replicates 10 mm equivalent SD mg mL CV NanoDrop 2000 0 03 to 300 0 1 mg mL to 400 mg mL 0 10 10 mg mL 0 10mg mL gt 10 mg mL 2 NanoDrop 2000c 0 003 to 300 0 010 mg mL to 400 mg mL 0 10 10 mg mL 0 10mg mL gt 10 mg mL 2 NanoDrop 8000 0 03 to 75 0 1 mg mL to 100 mg mL 0 15 5 mg mL 0 15 mg mL gt 5 mg mL 2 5 NanoDrop 1000 0 03 to 75 0 1 mg mL to 100 mg mL 0 10 10 mg mL 0 10 mg mL gt 10mg mL 2 Denotes lower detection limit when using 10 mm path cuvette The detection limits will vary based upon the extinction coefficients associated for specific proteins Thermo Scientific NanoDrop Spectrophotometers Protein A280 Calibration Calibration Verification All NanoDrop spectrophotometers include a diagnostic application which allows the user to run a Calibration Check procedure to confirm that the instrument is working within specifications Wavelength Calibration Automatic e Wavelength calibrations using standard reference lines in the xenon flashlamp spectrum are automatically performed within the operating software e This verification ensures wavelength accuracy and does not require any action by the user Pathlength Verification User performed e Use CF 1 in conjunction with the Calibration Check diagnostic to verify that the pathlengths are within specification e The pathlengths used
13. e refer to the model specific user manual for more detailed instrument and software feature related information The patented NanoDrop sample retention system employs surface tension to hold 0 5 to 2 uL samples in place between two optical fibers Separate booklets for nucleic acid and protein colorimetric methods are also available For technical support please contact Thermo Fisher Scientific NanoDrop Products 3411 Silverside Road Bancroft Building Wilmington DE 19810 USA Toll free in US and Canada 1 877 724 7690 Phone 1 302 479 7707 Fax 1 302 792 7155 e mail nanodrop thermofisher com Wwww nanodrop com Thermo Scientific NanoDrop Instrument Serial Numbers NanoDrop 1000 S N S N_ NanoDrop 2000 S N S N_ NanoDrop 2000c S N S N NanoDrop 8000 S N S N_ Introduction Sample Retention Technology Thermo Scientific NanoDrop Spectrophotometers Protein A280 e Pipette 1 2 uL sample directly onto the measurement pedestal Tip 0 5 uL aliquots may be used for samples that have 10 mm equivalent absorbance values of 3 0 or higher gt 4 5 mg mL BSA NanoDrop 2000 2000c model only e Lower the sampling arm and initiate a spectral measurement using the software on the PC e Surface tension is used to hold samples in place between two optical fibers e Light from a xenon flashlamp passes through the top optical fiber down through the liquid column and is detected by the internal sp
14. ection on page 13 for additional information regarding molar vs mass concentration values The molar absorption coefficient of a peptide or protein is related to its tryptophan W tyrosin Y and cysteine C amino acid composition e At 280 nm this value is approximated by the weighted sum of the 280 nm molar absorption coefficients of the three constituent amino acids as described in the following equation nW x 5500 nY x 1490 nC x 125 e nis the number of each residue e Stated values are the amino acid molar absorptivities at 280 nm 11 iw 2 2 z a Calculations Absorptivity Thermo Scientific NanoDrop Spectrophotometers Protein A280 Absorptivity is wavelength specific for each protein and can be affected by buffer type ionic strength and pH e The best extinction coefficient value is one that is determined empirically using a solution of the study protein at a known concentration Tip When determining extinction coefficients for a specific protein it is important to use the same buffer as will be used for the general assay e Refer to the Pierce bulletin TR0006 2 for additional information regarding the use of Pierce standards to calculate a system specific extinction coefficient Protein sample concentrations are determined based on the absorbance at 280 nm the extinction coefficients determined by the selected sample type and a baseli
15. ectrometer e When the measurement is complete raise the sampling arm and wipe the sample from both the upper and lower pedestals using a dry lint free laboratory wipe Using this technology NanoDrop spectrophotometers have the capability to measure samples between 50 and 200 times more concentrated than samples measured using a standard 1 cm cuvette The Protein A280 method is applicable to purified proteins that contain Trp Tyr residues or Cys Cys disulphide bonds and exhibit absorbance at 280 nm This method does not require generation of a standard curve and is ready for protein sample quantitation at software startup Colorimetric assays such as BCA Pierce 660 nm Bradford and Lowry require standard curves and are more commonly used for uncharacterized protein solutions and cell lysates lt a zZ O gt Best Practices Cleaning Reconditioning Pedestal Assessment Droplet flattens out on unconditioned pedestal Thermo Scientific NanoDrop Spectrophotometers Protein A280 e An initial cleaning of both measurement surfaces with dH 0 is recommended prior to making the blank measurement Do NOT use a squirt or spray bottle to apply water or any other liquid to the surface of the instrument e Between measurements Wipe the sample from both the upper and lower pedestals with a clean dry lint free lab wipe e A final cleaning of both measurement surfaces with dH 0 is recomme
16. ention system are resistant to sample adherence making the use of dry lint free lab wipes very effective in removing the sample Q How do keep my sample from flattening out on the measurement pedestal A Use the NanoDrop PR 1 reconditioning compound as a rapid means of reconditioning the pedestals when the surface properties have been compromised and liquid columns break during measurement PR 1 kits are available through Thermo Fisher Scientific or your local distributor sovd Q What is the cause of negative absorbance values A A blank measurement was made either using a solution with more absorbance than the sample of interest or on a dirty pedestal Clean the pedestal and make a new blank measurement with a fresh aliquot of the appropriate buffer Q What sort of reproducibility should expect with NanoDrop spectrophotometers A Typically within 2 at the 1 mm pathlength Q How do check the accuracy of NanoDrop spectrophotometers A NanoDrop CF 1 Calibration Check Fluid should be used with the Calibration Check diagnostic in the instrument software CF 1 is prepared from the NIST potassium dichromate standard SRM935 in acidified reagent grade water 27 28 FAQs Thermo Scientific NanoDrop Spectrophotometers Protein A280 Q How do calibrate NanoDrop spectrophotometers A The Calibration Check diagnostic allows the user to confirm that the instrument is performing within specifications If the i
17. he top of the 280 mm peak most likely indicates detector saturation due to a highly concentrated sample Absorbance Suggestion 5 In cases like these the data will be erroneous and should not be used for downstream work Dilute the sample and remeasure 0 220 230 240 250 260 270 280 290 300 310 320 330 340 350 Wavelength nm 21 Thermo Scientific NanoDrop Spectrophotometers Protein A280 Troubleshooting Non reproducible results are usually due to issues with sample non homogeneity blanking on a dirty pedestal using the same aliquot for multiple measurements or column breakage Reproducibility Sample Heterogeneity e Sampling from non homogeneous solutions particularly when using small volumes may result in significant measurement deviation in the generated data Dirty pedestal e Clean and recondition the pedestal surfaces prior to the start of the measurement session Follow the suggestions in the Blank Measurements section on page 9 prior to making sample measurements Multiple Measurements e Use fresh aliquots for each pedestal measurement Tip Multiple measurements of the same aliquot may result in evaporation and increased sample concentration values Column Breakage e Visually check that a column is intact after the completion of the measurement If not refer to the Instrument Related Issues section for guidance Thermo Scientific NanoDrop Spectrophotometers Protein
18. imited to photocopy photograph magnetic or other record without our prior written permission Microsoft Windows Windows NT and Excel are either trademarks or registered trademarks of Microsoft Corporation in the United States and or other countries Adobe and Acrobat are trademarks of Adobe Systems Incorporated All other trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries NanoDrop is a trademark of Thermo Fisher Scientific Revised 11 2010 Thermo Scientific NanoDrop Spectrophotometers Protein A280 Contents Introduction Sample Retention Technology 4 5 Best Practices 6 Cleaning amp Reconditioning 6 Instrument amp Cuvette Orientation 7 Sampling Technique 8 9 Measurements Blank Measurements 9 Sample Measurements 10 Calculations 11 Absorptivity 11 Published Extinction Coefficients 13 Sample Type Options 15 Pathlength amp Concentration 17 Calibration 18 Calibration Verification 18 Standard vs Control 19 Troubleshooting 20 Common Reagents 20 Unusual Spectra 21 Reproducibility 22 Instrument Related Issues 23 Installation Errors 24 Connection Errors 25 Signal Errors 25 FAQs 26 Technical Support 29 Introduction Thermo Scientific NanoDrop Spectrophotometers Protein A280 This booklet is meant to provide some basic protein measurement support information for direct A280 methods relevant to Thermo Scientific NanoDrop 2000 2000c 8000 and 1000 spectrophotometers Pleas
19. mon for protein work than percent solution Tip The variation in reporting style underscores the importance of carefully reading stated values to be sure that the unit of measure is understood and applied correctly Additional information is available in bulletin TR0006 2 at www piercenet com Thermo Scientific NanoDrop Spectrophotometers Protein A280 Calculations Conversions The relationship between molar extinction coefficient Published Extinction molar and percent extinction coefficient 1 is Coefficients as follows Enota 10 1 x molecular weight of protein Example Assume you want to determine the 1 for a protein whose molar extinction coefficient is 43 824 M cm and a molecular weight of 66 400 daltons To determine the 1 for this protein rearrange the above equation as follows E1 E noar 10 MW 1 43 824 10 66 400 daltons E1 6 6 e To report concentrations in terms of mg mL an adjustment factor of 10 is used when using percent solution extinction coefficients To convert from g 100 mL to mg mL A 1 10 concentration in mg mL Example Assume you obtain an 280 nm absorbance reading of 5 8A for a protein sample relative to your reference To determine the calculated concentration of your sample mg mL refer to the equation below C mg mL 7 A El x 10 C mgmt 9 8 6 6 x 10 C nq 8 79 mg mL Tip The NanoDrop software automatically i
20. ncludes the factor of 10 when reporting protein concentrations The information presented above is for explanation purposes only Thermo Scientific NanoDrop Spectrophotometers Protein A280 Calculations There are six sample types options available for purified protein analysis and concentration A measurement using the A280 application on the NanoDrop models specified at the beginning of ZA this guide A description of each sample type is given below Representative Sample Types BSA e Assuming a MW 66 400 daltons the molar Protein concentrations are extinction coefficient at 280 nm for BSA is approximately calculated using the mass 43 824 Mcm extinction coefficient of 6 7 at 280 nm for a 1 i e 10 mg mL Bovine Serum Albumin solution IgG e Most mammalian antibodies i e immunoglobulins Protein concentrations are have protein extinction coefficients percent in the calculated using the mass range of 12 to 15 extinction coefficient of 13 7 at 280 nm for a 1 i 10 mg mL For a typical IgG with MW 150 000 daltons this IgG solution value corresponds to a molar extinction coefficient equal to 210 000 M cm Lysozyme e Molar extinction coefficient for egg white lysozyme Protein concentrations are ranges from 36 000 to 39 000 Mcm calculated using the mass extinction coefficient of 26 4 at 280 nm for a 1 i e 10 mg mL Lysozyme solution Calculations Sample Ty
21. nded after the last sample measurement Do NOT use a squirt or spray bottle to apply water or any other liquid to the surface of the instrument e Additional cleaning Use 3 uL of HCI instead of the dH 0 for cleaning when samples have dried on the pedestal Follow with a 3 uL aliquot of dH 0 e Detergents and isopropyl alcohol are NOT recommended cleaning agents as they may uncondition the pedestal measurement surfaces If a solution containing detergents or alcohol is used follow with 3 5 uL of dH 0 Use the NanoDrop Pedestal Reconditioning Compound PR 1 as a rapid means of reconditioning the pedestals when the hydrophobic surface properties have been compromised and liquid columns break during measurement 1 Open the vial containing PR 1 and use the applicator provided in the kit to remove a pin head sized amount of the compound 2 Apply a very thin even layer of PR 1 to the flat surface of the upper and lower pedestals Wait 30 seconds for the PR 1 to dry 3 Fold a clean dry lint free lab wipe into quarters and remove the PR 1 by rubbing the surface of the upper and lower pedestals until no additional dark compound residue shows on the lab wipe To check the effectiveness of the reconditioning pipet a 1 uL aliquot of dH 0 onto the lower measurement pedestal and visually verify that the water beads up Droplet beads up on properly conditioned pedestal Thermo Scientific NanoDrop Spectrophotometers Protein A28
22. ne correction e The correction normalizes for any baseline offset attributable to light scattering artifacts The default wavelength for the baseline normalization is 340 nm Tip The user may manually enter a different wavelength to be used for the baseline normalization when using the NanoDrop 2000 2000c Calculations Published Extinction Coefficients Thermo Scientific NanoDrop Spectrophotometers Protein A280 Absorption coefficients i e extinction coefficients for many proteins have been compiled from the literature and reported in various handbooks These values provide sufficient accuracy for most routine laboratory applications that require an assessment of protein concentration Most sources report extinction coefficients for proteins measured at or near a wavelength of 280 nm in phosphate or other physiologic buffer Published Values Some resources provide coefficient values for specific proteins as the wavelength dependent molar absorptivity coefficient or extinction coefficient with units of Mcm e Some resources provide coefficient values for specific proteins as 1 1 g 100 mL solutions measured in a 1 cm cuvette These values can be understood as percent solution extinction coefficients 1 having units of g 100 mL cm instead of Mcm e Still other sources provide protein absorbance values for 0 1 i e 1 mg mL solutions as this unit of measure is more convenient and com
23. noDrop Product Technical Support Team is available between 9am and 5pm EST For technical support outside of the US and Canada please contact your local Thermo Scientific NanoDrop products distributor Additional technical information is available at www nanodrop com Thermo Fisher Scientific NanoDrop Products 3411 Silverside Road Bancroft Building Wilmington DE 19810 U S A Toll free in US and Canada 1 877 724 7690 Phone 1 302 479 7707 Fax 1 302 792 7155 E mail nanodrop thermofisher com www nanodrop com 29 oO iz O f N 3 cs Thermo Scientific NanoDrop Spectrophotometers Protein A280 Additional Notes ssDNA 33 ng cm pL emolar 10 1 x molecular weight of protein Kh x A iL a dsDNA 50 ng cm uL c A cb 1 Abs 1 mg mL Thermo Scientific NanoDrop Spectrophotometers Protein A280
24. nstrument requires recalibration contact Technical Support Outside of the US and Canada please contact your local NanoDrop products distributor Q Where is the data stored A The NanoDrop 2000 2000c software allows the user to save a workbook twbk at a location of the user s preference for recording measurements The default data storage location is in the My Documents folder The NanoDrop 8000 and the NanoDrop 1000 models automatically archive all measurement data in a folder on the C drive Refer to the model specific user manual for additional details A PDF version of each manual may be found at www nanodrop com Q Is the flash lamp continuously on or is it on only when performing a measurement A The lamp is on only during measurements Q Are there solvent restrictions A Yes Do not use hydrofluoric acid on the pedestal as it may etch the quartz optical fiber Most other laboratory solvents typically used in life science labs including dilute acids are compatible with the pedestal as long as they are immediately wiped off upon the completion of the measurement Tip The use of volatile solvents for sample measurement may result in erroneous data due to the rapid evaporation of the 1 2 uL sample volume Technical Support Thermo Scientific NanoDrop Spectrophotometers Protein A280 For additional assistance please contact us at 1 877 724 7690 or send an email to nanodrop thermofisher com The Thermo Scientific Na
25. pe Options Thermo Scientific NanoDrop Spectrophotometers Protein A280 Other Sample Types 1 Abs 1 mg ml A general reference setting based on a 0 1 i e 1 mg mL protein solution producing an Absorbance at 280 nm of 1 0 A where the pathlength is 10 mm or 1 cm e Assume 1 10 if no extinction coefficient information exists for a protein or protein mixture of interest and a rough estimate of protein concentration is required for a solution that has no other interfering substances e Most protein extinction coefficients 1 range from 4 0 to 24 0 Although any given protein can vary significantly from 1 10 the average for a mixture of many different proteins will likely be approximately 10 Tip This option is useful when measuring a protein solution for which no absorptivity information is available Other protein MW The relationship between molar extinction coefficient molar and percent extinction coefficient percent is as follows molar 10 percent x molecular weight of protein e Although the label of the field where one enters the extinction coefficient differs between NanoDrop models e g 1000 or x1000 the value entered would be the same For example for a protein with a molar extinction coefficient of 210 000 M cm enter 210 in the window for all model types e Enter the molecular weight in kilodaltons in the MW kDal field
26. s include purified proteins a Proteins and Labels module for labeled antibodies and other protein incorporating fluorescent labels or intrinsic fluorescence In addition colorimetric assays such as the BCA Bradford Modified Lowry and Pierce 660 nm Protein assay are easy and quick to run Custom methods may also be set up via the Method Editor on the NanoDrop 2000 2000c to analyze proteins including peptides at 205 nm available in the NanoDrop 2000 2000c operating software Q am using a colorimetric method e g Bradford BCA etc to determine my protein concentration Can measure my sample using the A280 application on NanoDrop spectrophotometers A Yes The A280 application is most applicable to purified proteins Colorimetric assays such as BCA Pierce 660 nm Bradford and Lowry are generally used for uncharacterized protein solutions and cell lysates If you are using a colorimetric assay now it is recommended that you continue to do so Q What are the sample size requirements when using NanoDrop spectrophotometers A We recommend using a 2 uL sample size for pedestal based protein measurements Proteins and or protein buffers may alter the surface tension properties of the solution and using the larger sample size is recommended to ensure proper column formation Q When is a 0 5 pL volume sufficient A The small sample volume option is available when samples have 10 mm equivalent absorbance values of 3 0 or higher gt
27. sample quality 10mm Absorbance 10mm Absorbance Oo ap a a ees 220 230 240 250 260 270 280 290 300 310 320 330 340 350 o 230 240 250 260 270 280 290 300 310 320 330 340 350 Wavelength nm Wavelength nm High concentration BSA sample Low concentration BSA sample Tip Refer to the Troubleshooting section on page 20 for more information Calculations Absorptivity Thermo Scientific NanoDrop Spectrophotometers Protein A280 The information in this section is a summary of the information presented in the Thermo Scientific Pierce products technical support bulletin TR0006 2 Please refer to www piercenet com for additional information or to reach Pierce technical support Beer s Law states that molar absorptivity is constant and the absorbance is proportional to concentration for a given substance dissolved in a given solute and measured at a given wavelength A e b c e A is the absorbance value A e is the wavelength dependent molar absorptivity coefficient or extinction coefficient with units of liter mol cm e bis the path length in centimeters e cis the analyte concentration in moles liter or molarity M Solving the expression of Beer s Law for concentration yields the following equation c A eb e Dividing the measured absorbance of a protein solution by the molar extinction coefficient yields the molar concentration of the protein solution Refer to the Published Extinction Coefficients s
28. splayed and the user visually confirms that the liquid column is forming perform a calibration check If the instrument is out of calibration contact Technical Support Outside of the US and Canada please contact your local NanoDrop products distributor 23 Troubleshooting Installation Errors Thermo Scientific NanoDrop Spectrophotometers Protein A280 Usually an installation failure is the result of an unsuccessful installation of the device drivers e Verify system specifications meets published requirements e Verify that user has full Administrator access to the software and data folders and that the use of USB devices is acceptable e Verify that the instrument is receiving power e Verify driver installation using the Device Manager 1 Locate the My Computer icon on the desktop or access through the Windows Start menu Right click on My Computer 2 Highlight and select Manage 3 Click on Device Manager in the left pane 4 Locate the NanoDrop device folder from the list displayed in the right pane and click on the plus sign to open Tip Yellow exclamation points or question marks associated with either a NanoDrop or an unknown device indicate drivers did not install properly 5 Highlight and delete the questionable device 6 Unplug the USB cable from the computer and the power cord from the instrument Wait 10 seconds then reconnect beginning with the power cord If the error persists
29. to determine if your buffer exhibits significant absorbance in the region of interest 1 Perform the Cleaning and Reconditioning procedures outlined in the Best Practices section on page 6 2 Open the A280 application Load an aliquot of dH 0 onto the lower measurement pedestal and lower the sampling arm 3 Click Blank After the measurement is complete use a dry lint free lab wipe to remove the water from both the top and bottom measurement surfaces 4 Pipette an aliquot of the sample buffer onto the pedestal lower the arm and click Measure The result should be a spectrum that varies no more than 0 04 absorbance 10 mm absorbance equivalent from the baseline at 280 nm If not consider using a colorimetric method to quantitate the protein samples 1 04 20 E a4 10mm Absorbance gt S K oR 10mm Absorbance 0 0 o 220 230 240 250 260 270 280 290 300 310 320 330 340 350 30 240 240 250 260 270 280 290 300 310 320 330 340 350 Wavelength nm Wavelength nm Example spectrum of buffer suitable Example spectrum of buffer unsuitable for A280 protein quantitation for use with A280 protein quantitation 5 If the buffer is compatible with the A280 method load a fresh aliquot of the buffer onto the lower measurement pedestal and lower the sampling arm Click Blank 6 After the measurement is complete use a dry lint free lab wipe to remove the buffer from both the top and bottom measurement surfaces
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