Protein Express Assay User Guide
Contents
1. ssssssssssrsssssssrsrrsssrsrnrnnnrrr nn nn n rr nn nn nn 31 Chip Well Aspiration Using a Vacuum s s ssssssssssrrssssssrrnsnnrsrrrnn nn k r nn n RKA RDR R NRK RN RR RR nn 32 Customer Technical Support s sssssssssssssssrrssssssrnnnnnsarnnnnnnkA RR Rn RAR R REAR RAA RR RR RAR RR nn 33 Licenses and Rights Of Use ssssssssssssssssssssssrsnnrrrsnrrrn ss nn kn non senses vive ceveueeseuncents 34 PN CLS140156 Rev D Protein Express Assay User Guide Specifications Assay Specifications Table Specifications 3 1 Assay Specifications Sizing Range P100 Assay 14 kDa 100 kDa P200 Assay 14 kDa 200 kDa Sizing Resolution 10 difference in molecular Concentration weight Sizing Accuracy 20 Linear Concentration Range 5 0 2000 ng uL Maximum Total Protein 10 mg mL Quantitation Reproducibility 30 CV up to 120 kDa Above 120 kDa quantitation is not specified Prep Sample Capacity per Chip 400 samples four 96 well plates or one 384 well plate a Resolution is defined as the height of the valley between two peaks to be no more than 50 of the maximum peak height Actual separation performance can depend on the sample and application Sample Conditions Table 2 Sample Conditions Buffers Salts and Additives Refer to Compatible Buffers Salts and Additives on page 31 for compatibility with specific buffers salts and additives If2. Touch the Unload Chip button on the Home screen For GXII press the Chip button Protein Express Assay User Guide Preparation Procedures 12 Chip Status Type HT Protein Express Chip Reagents N A Chip Expiration Jan 1 2020 Chip Life 400 Samples Left Status HOH CC AA ees C SR X OOO H H H OQ X K x rg x H Unload Unload Plate Chip Figure 5 Home screen 4 Insert the chip into the LabChip GXII Touch instrument Figure 6 and close the chip door securely For GXIl release the latch insert the chip latch the chip cartridge and push in the cartridge Figure 6 Chip in the LabChip GXII Touch instrument 5 Touch the Load Plate button on the Home screen Figure 5 to retract the sample plate and move the sipper to the Buffer Tube For GXIl press the Eject button Note Do not keep the chip door open for any length of time Dye is sensitive to light and can be photobleached PN CLS140156 Rev D Protein Express Assay User Guide Preparation Procedures 13 Running the Assay Note Chips can be primed independently from running assays on the LabChip GXII Touch instrument Touch the Prime button on the Home screen Figure 5 Select the desired assay from the Assay drop down list see Figure 8 Touch the Prime button on the Chip Priming screen Make sure the Buffer Tube is placed on the instrument Chip Status Assay Type HT Protein Express ur Antibody Analysis 200 High S
3. Assay Lower Marker 11 13 5 seconds e Upper Ladder Protein on the first plate 23 32 seconds e Upper Ladder Protein on the third plate 20 29 seconds Possible causes 1 Incorrect Gel Dye ratio Migration time is sensitive to dye concentration and peaks will migrate too fast or too slow if the dye concentration in the gel is too low or too high respectively Note Excess dye within the separation channel will slow down migration and less dye in the separation channel will make peaks migrate faster 2 Particulates from the samples may be clogging the separation channel this will slow down migration 3 Gel Dye was not primed properly into the chip What to do 1 Prepare a fresh Gel Dye solution Wash and reprime the chip with the new Gel Dye mixture See LabChip Kit Essential Practices on page 25 for instructions on how to wash and reprime the chip PN CLS140156 Rev D Protein Express Assay User Guide 2 Troubleshooting 24 If fast or slow migration is observed repeatedly on a new chip contact technical support to arrange return of the chip to PerkinElmer Please send a data file showing the failure along with the return request Minimize the loading of particulates in the sample by performing a centrifuge spin of the sample plate e g 3000 rcf for 5 minutes and or ensuring the Sip 4 mm plate type is selected in the Select Wells screen before starting a new run The debris may be flushed out of the chip by washing
4. JK JR TR TJ JAMIN MM NV vv vv vv Ladder Tube Buffer Tube Figure 1 Locations of the Buffer Tube and Ladder Tube in the GXII Touch instrument Preparing the Chip 1 Allow the chip to come to room temperature 2 Use a pipette tip attached to a vacuum line to thoroughly aspirate all fluid from the chip wells see Figure 2 For more details on how to set up a vacuum line see page 32 3 Each active chip well 1 2 3 4 7 8 9 and 10 should be rinsed and completely aspirated twice with water Milli Q or equivalent Do not allow active wells to remain dry 4 If any water spills onto the top or bottom of the chip surfaces during rinsing aspirate using the vacuum line DO NOT run the tip over the central region of the detection window Use the provided Detection Window Cleaning Cloth to clean the chip detection window PN CLS140156 Rev D Protein Express Assay User Guide Preparation Procedures 10 Figure 2 Using a vacuum to aspirate the chip wells is more effective than using a pipette See page 32 for more details 5 Using a reverse pipetting technique add Gel Dye solution to chip wells 3 7 8 and 10 as shown in Figure 3 High throughput or Figure 4 Low throughput 1 2 9 3 4 OO LB 4 OL XO marker gel Dye Destain Marker gel Dye Destain Figure 3 Reagent placement Figure 4 Reagent placement for High throughput up to for Low throughput up to 48 384 samples
5. Milli Q or equivalent to clean the electrodes and the O rings using a circular motion If the O rings stick to the chip or a pressure leak is detected perform the more extensive monthly cleaning procedure 2 Monthly PN CLS140156 Rev D a To reduce pressure leaks at the chip interface clean the O c rings frequently Remove the O rings from the top plate of the chip interface on the LabChip GXII Touch or GXII instrument Soak O rings in water Milli Q or equivalent for a few minutes Clean the O ring faces by rubbing between two fingers Wear gloves To reduce the occurrence of current leaks clean the chip interface frequently Clean the top plate of the chip interface using the provided lint free swab dampened with water Milli Q or equivalent Allow the O rings and chip interface to air dry Reinsert the O rings into the chip cartridge Protein Express Assay User Guide Results 18 Results Protein Express Ladder Result The electropherogram of a typical Protein Express ladder is shown in Figure 12 Peaks to the right of the lower marker and system peaks in order of increasing migration time correspond to proteins of increasing size i e 15 9 kDa 20 4 kDa 28 9 kDa 48 4 kDa 68 4 kDa and 119 2 kDa respectively Time sec Figure 12 Protein Express ladder electropherogram PN CLS140156 Rev D Protein Express Assay User Guide Troubleshooting 19 Troubleshooting Note Some of the data examp
6. VR ecco Type HT Protein Express Chip Reagents N A 333 e D Chip Expiration Jan 1 2020 ados Chip Life 400 Samples Left Run Parameters Operator IM 4 HS Assay HT Antibody Analysis 200 High Sensitivity F x i 7s 5 X Data Path C Wrogram Files x86 PerkinElmer G OOOC OC OOO Ig LabChip GX Touch Data Plate Name BioRad 96 HSP 96xx Sip 4 mm H 000808 K Barcode N A Plate Cycles 1 Auto Export No Sample Saver No Random Selection No 090090 Figure 11 Starting a run Storing the Chip After use the chip must be cleaned and stored in the chip container 1 Place the chip into the plastic storage container The sipper should be submerged in the fluid reservoir 2 Remove the reagents from each well of the chip using vacuum 3 Each active well 1 2 3 4 7 8 9 and 10 should be rinsed and aspirated twice with water Milli Q or equivalent 4 Add 120 uL water Milli Q or equivalent to the active wells 5 Cover the wells with Parafilm to prevent evaporation and store the chip at room temperature until next use The chip must be used to its lifetime to the total number of 400 samples within 30 days of analyzing the first plate of samples Chip Cartridge Cleaning 1 Daily a Inspect the inside of the chip cartridge and O rings for debris PN CLS140156 Rev D Protein Express Assay User Guide Preparation Procedures 17 b Use the provided lint free swab dampened with water
7. and re priming the chip See LabChip Kit Essential Practices on page 25 for instructions on how to wash and reprime the chip Check the O rings on the top surface of the chip interface and clean if necessary Symptom High baseline fluorescence e g greater than 1000 counts Possible causes 1 The destain wells 2 and 9 do not contain Destain solution gel matrix with no dye 2 The destain wells 2 and 9 may have been contaminated with dye either because the well was improperly flushed after priming or because dye was mistakenly pipetted into the well What to do 1 Prepare a fresh Destain solution Wash and reprime the chip with the new Destain solution See LabChip Kit Essential Practices on page 25 for instructions on how to wash and reprime the chip Symptom Lower than expected signal for ladders and samples Possible causes 1 Improper SDS concentration in Gel Dye matrix What to do 1 Ensure that Dye Concentrate is completely thawed and mixed PN CLS140156 Rev D Prepare a fresh Gel Dye solution Wash and reprime the chip with the new Gel Dye mixture See LabChip Kit Essential Practices on page 25 for instructions on how to wash and reprime the chip Protein Express Assay User Guide LabChip Kit Essential Practices 25 LabChip Kit Essential Practices To ensure proper assay performance please follow the important handling practices described below Failure to observe these guidelines
8. any other purposes contact Life Technologies Corporation 5791 Van Allen Way Carlsbad CA 92008 USA or outlicensing lifetech com Rights of Use The chip and reagents supplied with this kit are sold with limited rights of use The chip may only be used with the specific quantity of reagents supplied with this kit The purchaser has no right or license to refurbish reuse remanufacture or otherwise use the chip with any other reagents than those specifically supplied in this kit For more information on the terms and conditions of use of these chips and reagents please read your LabChip GX Touch or LabChip GXII User Guide and refer to the applicable label license The reagent kit contains materials that are provided under a license from Life Technologies Corporation for research use only PerkinElmer LabChip and the LabChip logo are registered trademarks of PerkinElmer Inc and or its parent affiliates and or subsidiary companies collectively PerkinElmer The PerkinElmer logo is a registered trademark of PerkinElmer Inc All other trademarks and registered trademarks are the property of their respective holders 2014 2015 PerkinElmer Inc http www perkinelmer com PN CLS140156 Rev D Protein Express Assay User Guide I PerkinE PerkinElmer Inc 68 Elm Street Hopkinton Massachusetts 01748 U S A TEL 508 435 9500 FAX 508 435 3439 http www perkinelmer com
9. but requires a larger amount of sample Slightly lower resolution may be observed Protein Express 200 For sizing of proteins in the 14 kDa to 200 kDa range Protein Express 200 High Sensitivity For sizing of proteins in the 14 kDa to 200 kDa range Greater sensitivity but requires a larger amount of sample Slightly lower resolution may be observed Antibody Analysis For analyzing Ab samples Same choices of assays as in Protein Express 3 Inthe Setup Run tab select the operator name the option to read barcode the destination of the file the inclusion of sample names expected peaks and excluded peaks and the filename convention Select Auto Export to export results tables automatically Figure 10 Touch the green arrow Status PN CLS140156 Rev D Run Setup Run Start Run Operator Read Plate Barcode Data Path C Program Files x86 PerkinElmer LabChip GX Touch Data Browse Default MPi Fil PerkinEl hi Touch Ee Iv Copy to C Program Files x86 PerkinElmer LabChip GX Touch DataCopy En DRA VALS Daily Sub Directory Auto Export Data File Name Labchip 2014 06 07 07 55 10 gxd File Prefix Project name Labchip Computer Name Barcode y Date vV Time Advanced Settings Sample Names File Expected Peak File Excluded Peak File Figure 10 Run setup screen Protein Express Assay User Guide Preparation Procedures 16 4 Touch click Start to begin the run Run Select Wells Setup Run Start Run Chip Status
10. may void the LabChip Kit product warranty Note It is important to keep particulates out of the chip wells channels and capillary Many of the following guidelines are designed to keep the chips particulate free For assay and instrument troubleshooting refer to the Software Help file or call PerkinElmer Technical Support at 1 800 762 4000 General PN CLS140156 Rev D Allow the chip sample plate and all reagents to equilibrate to room temperature before use approximately 20 to 30 minutes Clean the O rings in the chip interface weekly and the electrodes daily Refer to the Instrument Users Guide Maintenance and Service section for procedures Avoid use of powdered gloves Use only non powdered gloves when handling chips reagents sample plates and when cleaning the instrument electrodes and electrode block Calibrate laboratory pipettes regularly to ensure proper reagent dispensing Only the PerkinElmer supplied Detection Window Cleaning cloth can be used on the chip to clean the detection window Water used for chip preparation procedures must be 18 megohm 0 22 um filtered water Milli Q or equivalent Using the Reverse Pipetting Technique on page 26 will help avoid introducing bubbles into the chip when pipetting the gel PerkinElmer Inc warrants that the LabChip Kit meets specification at the time of shipment and is free from defects in material and workmanship LabChip Kits are warranted for 60 days f
11. samples Note Up to 48 samples can be analyzed in LT mode 6 Using a reverse pipetting technique add Destain Solution orange cap to chip wells 2 and 9 as shown in Figure 3 High throughput or Figure 4 Low throughput PN CLS140156 Rev D Protein Express Assay User Guide 7 Preparation Procedures 11 If the chip will be used to analyze multiple 96 well plates or will be in use for up to 8 hours add 120 uL of Protein Express Lower Marker green cap to chip well 4 If the chip will only be used to analyze one 96 well plate or a partial plate and then stored for future use the marker volume can be reduced to 50 uL Make sure the marker volume is pipetted accurately If there is not enough marker in chip well 4 the marker will deplete and will not be added to subsequent samples on chip Data collected without marker peaks cannot be analyzed by the software Make sure the rims of the chip wells are clean and dry IMPORTANT Ensure chip well 1 waste well is empty before placing the chip into the instrument Inserting a Chip into the LabChip GXII Touch or GXII Instrument j PN CLS140156 Rev D Check that the sample plate Buffer Tube and Ladder Tube are placed on the instrument properly Remove the chip from the chip storage container and inspect the chip window Clean BOTH sides of the chip window with the PerkinElmer supplied Detection Window Cleaning cloth dampened with a 70 isopropanol solution in DI water
12. to a house vacuum line B vacuum line with trap Figure 20 Replacing the disposable pipette tip PN CLS140156 Rev D Protein Express Assay User Guide Customer Technical Support 33 Customer Technical Support PerkinElmer Inc 68 Elm Street Hopkinton MA 01748 1668 PerkinElmer Technical Support Phone USA Toll Free 800 762 4000 Phone Worldwide 1 203 925 4602 Fax 1 203 925 4602 Email global techsupport perkinelmer com Internet www perkinelmer com For additional assay and instrument troubleshooting refer to the Software Help file PN CLS140156 Rev D Protein Express Assay User Guide Licenses and Rights of Use 34 Licenses and Rights of Use Label Licenses This product is provided under an intellectual property license from Life Technologies Corporation The purchase of this product conveys to the buyer the non transferable right to use the data for internal research and training purposes solely in combination with PerkinElmer Inc instrumentation and not to generate revenue which may include but is not limited to use of the product 1 in manufacturing or in quality assurance or quality control of a commercial product 2 to provide a service information or data for a fee or other consideration 3 for therapeutic or prophylactic purposes 4 for diagnostic use and 5 for resale whether or not such items are resold for use in research For information on purchasing a license to this product for
13. water Milli Q or equivalent water used for chip preparation e Replace the buffer used for sample and reagent preparation e Use a0 22 micron filter for all water and buffers used for chip sample and reagent preparation e Spin down sample plate to pellet any particulates Symptom Humps in several electropherograms which do not correspond to sample data 1250 1000 ov o Fluorescence T o mhor 1 4 6 8 9 1 1 20 25 30 35 40 45 50 55 60 Time seconds Figure 15 Humps in several electropherograms PN CLS140156 Rev D Protein Express Assay User Guide Troubleshooting 23 Possible causes 1 Electrode 7 is dirty and has contaminated the Gel Dye mixture in well 7 2 High concentrations of detergent in the sample buffer can sometimes cause humps in the electropherogram What to do 1 Before restarting the run clean electrode 7 Remove the chip and follow the electrode cleaning procedure We recommend using the provided swab and isopropanol to manually clean electrode 7 2 Lower the detergent concentration in the sample see Compatible Buffers Salts and Additives on page 31 Symptom Peaks migrating much faster or slower than expected Note Some migration time variance between chips or within a plate is considered normal chip performance All chips are QC tested at PerkinElmer prior to shipment Normal migration time windows for the markers are e Protein Express
14. 60499 Touch HT or GXII Protein Express LabChip for use with GXII CLS138950 Touch 24 PN CLS140156 Rev D Protein Express Assay User Guide Safety Warnings and Precautions 5 Safety Warnings and Precautions WARNING A For Research Use Only Not recommended or intended for diagnosis of disease in humans or animals Do not use internally or externally in humans or animals CAUTION We recommend that this product and components be handled only by those who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice As all chemicals should be considered as potentially hazardous it is advisable when handling chemical reagents to wear suitable protective clothing such as laboratory overalls safety glasses and gloves Care should be taken to avoid contact with skin or eyes In case of contact with skin or eyes wash immediately with water WARNING AA e Dye Solution contains DMSO Avoid contact with skin and eyes e Dye Solution contains SDS Avoid inhalation and contact with skin and eyes e Wash Buffer and Sample Buffer contain LDS Avoid inhalation and contact with skin and eyes e Gel Matrix contains Methyl urea Avoid contact with skin and eyes PN CLS140156 Rev D Protein Express Assay User Guide Preparation Procedures 6 Preparation Procedures Additional Items Required 0 6 mL centrifuge tubes and or 96 well plates fo
15. I Perkin Protein Express Assay User Guide For LabChip GXII Touch and LabChip GXII The Protocols defined in this User Guide apply to all of the LabChip instrument products running the Protein Express Assay The LabChip GXII Touch is used for general visual reference in most examples in this guide CE Copyright 2014 2015 PerkinElmer Inc All rights reserved PerkinElmer is a registered trademark of PerkinElmer Inc All other trademarks are the property of their respective owners PN CLS140156 Rev D Contents Specifica CINE tos s2isse Facets si saa a Ga eased a eet cot de a dae 3 Assay Specificati nS scar oss cere dal oas a dee Beacon cones datan bad a E 3 Sample Conditions srein a Baer pee ERa Ea E E E e Flo ker AK AR 3 Kit Contents sesen eE aaa a sak a ea aaae 4 Safety Warnings and PreCaUtiONS sssssssssrsssrsssssrrrsrsssrrrrrann kkr nn nn RAR RNE RR RKA RR RAR NAR 5 Preparation Procedures s ssssssssssasssssrssssssssssnnnannnnn Han n kk nan ARR R ARR AR ARR RR ARR ARENA RER KRK Rn nn 6 Additional Items Required ovsessssssssrsssssrrssrrsrrrrerrrrornnsrsrrrnn rr rr RKRR AR Ron RR KKR KRK ERK KR DER Krona 6 Preparing the Gel Dye Solution molnmuseeserssrrrressrrrrrerorrrrrerrrnrrrrrrn rr rr rer ran rr rr rer RR rr ora na 6 Preparing the Sample Denaturing Solution mesmesesssrrersasrrrrrrnrrrrrerrrnrrrrrr nns rr rerna na 7 Preparing the Protein Samples and Ladder missmrsrersrrsrrerersrrrrrrsnrrrrrrrrnrrrrrr rr r rr ran
16. arations in low throughput mode whichever comes first Protein Express Assay User Guide Chips LabChip Kit Essential Practices 27 Repriming Chips Washing Chips e Touch the Unload Chip button on the Home screen to open the instrument door For GXII press the Chip button Place the chip into the instrument e Close the chip door securely and choose the corresponding assay For GXIl latch the chip cartridge push the chip cartridge in and press the Eject button e Touch the Prime button on the Home screen select the assay and touch the Prime button to reprime the chip For GXII run the desired assay The chip is primed at the start of the run Important Note Wash chips only with water Milli Q or equivalent Use of any other reagents including Wash Buffer is likely to cause even more artifacts in subsequent data Notes Some protein samples may have components which produce data with extra peaks spikes or other artifacts When these artifacts are present washing chips on the LabChip GXII Touch or GXII immediately before the next use can often restore data quality Chips should only be washed on the LabChip GXII Touch or GX immediately before they are prepared with fresh reagents and primed on the instrument Chips should not be washed and left with water in the chip channels for any extended period of time For most protein samples the only chip cleaning protocol that is required is to rinse and aspirate
17. ensitivity Chip Reagents N A Chip Expiration Jan 1 2020 Chip Life 400 Samples Left Status Buffer Figure 7 Chip priming screen 1 Touch click the Run button see Figure 5 on page 12 PN CLS140156 Rev D Protein Express Assay User Guide Preparation Procedures 14 2 Select the appropriate assay type see Figure 8 plate name well pattern and whether to read wells in columns or rows Select number of times each well is sampled under Adv Settings Figure 9 Touch the green arrow HT Antibody Analysis 200 High Sensitivity HT Antibody Analysis 200 High Sensitivity HT Antibody Analysis 200 HT Pico Protein Express 100 HT Pico Protein Express 200 HT Protein Express 100 High Sensitivity HT Protein Express 100 HT Protein Express 200 High Sensitivity HT Protein Express 200 Figure 8 The Assay list Run elect Well Setup Run Start Run Assay Folder C Program Files x86 PerkinElmer LabChip GX Touch Assay Select Assay Select Plate Type f HT Antibody Analysis 200 High Sensitivity v BioRad 96 HSP 96xx Sip 4 mm TRE veeeee 3 0 of 96 selected Figure 9 Selecting wells PN CLS140156 Rev D Protein Express Assay User Guide Preparation Procedures 15 For Protein Express assays appropriate assay types are Protein Express 100 For sizing of proteins in the 14 kDa to 100 kDa range Protein Express 100 High Sensitivity For sizing of proteins in the 14 kDa to 100 kDa range Greater sensitivity
18. h sample well or sample tube Vortex the sample tubes if used for a few seconds If using a plate a pipettor or plate shaker can be used to mix the water with the samples 8 Add 120 uL of water Milli Q or equivalent to the Protein Express Ladder Vortex the ladder mixture for a few seconds to achieve good mixing 9 Spin the Protein Express Ladder and sample tubes if used for 15 seconds using a mini centrifuge 10 If the samples are prepared in tubes transfer 44 uL of each sample onto a 96 well plate 11 Spin the sample plate to eliminate bubbles and move the fluid to the bottom of the wells 12 Place the sample plate onto the instrument s plate holder 13 Transfer 120 uL of prepared ladder to the provided 0 2 mL Ladder Tube Ensure there are no air bubbles in the Ladder Tube 14 Insert the Ladder Tube into ladder slot on the LabChip GXII Touch or GXII instrument Preparing the Buffer Tube 1 Add 750 uL of Protein Express Wash Buffer purple cap to the 0 75 mL Buffer Tube provided with the reagent kit Ensure there are no air bubbles in the Buffer Tube 2 Insert the Buffer Tube into the buffer slot on the LabChip GXII Touch or GXII instrument PN CLS140156 Rev D Protein Express Assay User Guide Preparation Procedures 9 Note Replace the Buffer Tube with a freshly prepared tube every 8 hours when the chip and instrument are in use Buffer Tube Ladder Tube vv vw ww ww Np V Sample JR OR JK OK JE JE JK
19. he Gel Dye mixture has an even blue color before transferring to the spin filter Insufficient mixing of gel and dye will cause inconsistent assay results PN CLS140156 Rev D Protein Express Assay User Guide Preparation Procedures 7 Transfer the solution into one of the spin filters provided with the reagent kit Centrifuge the solutions in the spin filter at 9300 rcf for 7 5 minutes at RT Remove the filter in the tube and discard Label and date the tube and store in the dark until the chip is to be prepared The volume of Gel Dye solution prepared is the required amount for one HT High Throughput or two LT Low Throughput chip preps If preparing additional solution for future use store the remaining solution in the dark at 4 C and use within 3 weeks Preparing the Sample Denaturing Solution 1 Pipette 700 uL of Protein Express Sample Buffer white cap into a 2 0 mL centrifuge vial If samples need to be reduced add 24 5 uL of BME or 1 M DTT or 3 75 uL of 100 mM TCEP Vortex for 10 seconds This volume of sample buffer and reducing agent is sufficient to prepare 96 samples A smaller volume can be prepared if running fewer than 96 samples Preparing the Protein Samples and Ladder Note Samples can be prepared in either a 96 well or 384 well PCR plate or in 0 6 mL microcentrifuge tubes and subsequently pipetted into a plate Procedures for both are described here 1 PN CLS140156 Rev D For each sa
20. hould not be exposed to an open environment for long periods of time Use care in chip handling to prevent sipper damage Damage to the sipper can result in inconsistent sampling Avoid exposing the chips to dust by keeping them in a closed environment such as in the chip container or in the instrument before and after chip preparation Protein Express Assay User Guide Samples PN CLS140156 Rev D LabChip Kit Essential Practices 30 Chips can be prepared and left in the instrument for extended periods of time so that samples can be run as needed throughout the day PerkinElmer recommends the chip be re prepared after it has been idle for 8 hours but the chip can be used continually over an 8 hour work day as long as the maximum recommended idle time of 8 hours and total chip lifetime of 400 samples are not exceeded Prepared sample plates should be free of gas bubbles and particulate debris both of which may inhibit sipper flow Sample plates containing gas bubbles and or particulate debris should be spun down at 3000 rpm 1250 rcf prior to analysis Up to four 96 well plates 400 samples can be run with a single chip preparation when running the GXII Touch HT or GXII instrument Up to 48 samples can be run with a single chip preparation when running the GXII Touch 24 instrument Protein Express Assay User Guide Compatible Buffers Salts and Additives 31 Compatible Buffers Salts and Additives Table 6 Compatible Buffe
21. l fluid from chip well 4 using a vacuum line Protein Express Assay User Guide Troubleshooting 21 Ensure that chip well 4 is rinsed and completely aspirated twice with water Milli Q or equivalent e Add Marker Solution green cap to chip well 4 e Reinsert the chip back into the instrument e Restart the run 2 Perform a marker channel unclogging procedure by repriming the chip See LabChip Kit Essential Practices on page 25 for instructions on how to reprime the chip Symptom Ladder traces show up in the lanes following the ladders delayed sip L JA A b d E Abgned Time sex Figure 13 Small ladder peaks in sample well caused by delayed sip Possible causes 1 Separation channel overloaded with sample 2 Partial clog in the separation channel What to do 1 Lower the starting sample concentration 2 Reprime the chip See LabChip Kit Essential Practices on page 25 for instructions on how to reprime the chip PN CLS140156 Rev D Protein Express Assay User Guide Troubleshooting 22 Symptom Unexpected sharp peaks 600 Lower Upper marker marker Peak caused by particulates Fluorescence w o S 40 45 50 55 Aligned Time sec Figure 14 Unexpected sharp peak Possible causes Dust or other particulates introduced through sample or reagents What to do 1 Doone or all of the following Replace the 18 megohm 0 22 um filtered
22. les shown in this section were generated with assays other than the assay described in this user guide Symptom No ladder or sample peaks but marker peaks detected Note The lower marker peak height will most likely be greater than normal height Possible causes 1 Air bubble in sipper introduced during chip priming What to do 1 Reprime the chip See LabChip Kit Essential Practices on page 25 for instructions on how to reprime the chip Symptom Missing sample ladder and marker peaks Possible causes 1 Clog in sipper or marker channel of chip What to do 1 Reprime the chip See LabChip Kit Essential Practices on page 25 for instructions on how to reprime the chip Symptom Ladder detected but no sample peaks Possible causes 1 The sipper is not reaching the sample due to low sample volume in the well of the plate 2 If the missing sample peaks occurred only in a few wells of the plate check those wells for air bubbles 3 The sipper is not reaching the sample due to an incorrect capillary height setting or incorrect plate definition 4 Ifthe plate has been uncovered for some time sample evaporation might have occurred 5 Debris from the sample or sample prep is clogging the sipper What to do 1 Add more sample to the well PN CLS140156 Rev D Protein Express Assay User Guide Troubleshooting 20 Manually insert a larger volume pipette tip 100 uL into the sample well and dislodge the b
23. m line Replace fluid in the wells with freshly made reagents as described in Preparing the Chip on page 9 Do not let wells remain dry e Transfer 750 uL of Wash Buffer purple cap into a clean Buffer Tube Install into the instrument e Install the Ladder Tube sample plate and chip into the instrument and run the assay PN CLS140156 Rev D Protein Express Assay User Guide LabChip Kit Essential Practices 29 If air bubbles are not dislodged after a reprime apply a vacuum to the sipper Perform this by filling all active wells with 100 uL water Milli Q or equivalent Then suction the sipper with a vacuum line as shown in Figure 18 until droplets of fluid flow out from the sipper When suctioning the sipper be careful not to bend or break the sipper To facilitate this cut the end of the pipette tip attached to the vacuum line to widen the mouth Figure 18 Removing an air bubble or clog by suctioning the sipper with a vacuum line Other Considerations PN CLS140156 Rev D New chips should be stored refrigerated prior to first use After running the first set of samples chips must be stored at room temperature and used within 30 days Do not allow the liquid in the chip container to freeze as this may lead to poor chip performance Do not submerge the chip in any solution The entire chip surface must be thoroughly dry before use The sipper must be kept immersed in fluid at all times and s
24. mple to be analyzed pipette 7 uL of denaturing solution into the wells of a microtiter plate or into individual 0 6 mL microcentrifuge tubes Pipette 2 uL of each protein sample into the wells of the 96 well plate or microcentrifuge tube When finished cover the plate with foil to minimize evaporation If running the High Sensitivity Assay pipette 5 uL of each protein sample Allow the Protein Express Ladder yellow cap to warm up to room temperature for 15 20 minutes followed by gentle vortexing for 10 seconds Briefly spin the ladder vial Ensure no precipitate is visible in the solution If precipitate is present let the vial sit at room temperature for a little longer and then repeat the gentle vortex and spin Protein Express Assay User Guide Preparation Procedures 8 4 Pipette 12 uL of Protein Express Ladder into a microcentrifuge tube or into the well of the microtiter plate Do not add denaturing solution to the ladder 5 Denature samples and ladder at 100 C for 5 minutes Optimum denaturing conditions may vary by sample type 6 Tap or spin the sample plate to move the fluid to the bottom of the wells 7 Add 35 uL of water Milli Q or equivalent to each sample well or sample tube and mix by pipetting up and down few times Avoid creating air bubbles This step should not be done more than an hour before starting the assay If running the High Sensitivity assay add 32 uL of water Milli Q or equivalent to eac
25. n 7 Preparing the Buffer Tube mossressrrssrrrssrrssessrssrrrsrrrrnrrrsssn rss KRK DARK DRA KR SRK KKR KRK R RR KKR Kona 8 Preparing the Chip eosssesserrressrssrersrssrrrrrsnrrrrrenrrrrrrrr an Seen rr r RK K RAR KKR KRK RR KKR KKR RR KR Kon ann 9 Inserting a Chip into the LabChip GXII Touch or GXII Instrument 0 11 RUNNING TG ASSAY e te S58 eee das cals vn AAA AA aAA ON sede aa cs ant RAAS Aiaia SAN ARS 13 Storing the CHIP mehrdan nne ss ia EEE EO S ka Dede RER MARE ee aegis lye eee 16 Chip Cartridge Cleaning messneserssrrrerasrrsreenrrrrrerrrnrrrrrr nec guaiee seca Kr rr RR RKA K RR RR RR KK KSR rr non 17 PROS ULES aaps es bednnd seek eatin sea ns k Ki ns ne ss aSa ERNER deai eooo anadan Ea earan iaoiai 18 Protein Express Ladder Result mstmssesssesssssrsssrrsrsrrorsrsrsrrrrsrrrrrrrrrn rr ror srr KKR KR KRK KR Dn rn 18 Troubleshooting ocsosesanie spsnsvedess skin kens taut rn s nn NER AA NN N sa Dede es HANSA Ran ANDRE SRSRNN KRKA Na anes 19 LabChip Kit Essential PraCtiCeS s ssssssssssssssssssssrrrnsnsrrnrnnnnrrr nn nns k KKR RKA KAR RR RNA 25 Generali pirrur eee ae hea an aaae aaraa penaa E E ega EAA NE S er dehy 25 Reagen S den rs dens ivnpnde der s sekr Hake KRK DSS NEN A PAEA Men Snel KKR KRKA RR SR HONAN aan 26 GHIPS riren as DERES SAS EAA a ERAN n ARE DS a aaa 27 Samples Fe barns san baffled a seen ee Hen Eee aged sulin Sf bl Ses behav owas Malee RE sage Ceca bees 30 Compatible Buffers Salts and AdditiVeS
26. r denaturing protein samples e Means for heating samples to 100 C 96 well PCR instrument or heating block Note Avoid using non stick lab consumables They may induce unexpected or erratic assay results caused by surface treatments leaching into dye or gel components 18 megohm 0 22 um filtered water Milli Q or equivalent e 70 isopropanol solution in DI water e Reducing agents BME beta mercaptoethanol 1M DTT dithiothreitol or 100 mM TCEP Note Allow the chip and all reagents to equilibrate to room temperature before use approximately 20 to 30 minutes Preparing the Gel Dye Solution Notes The Dye solution contains DMSO and must be thawed completely before use After warming the dye at room temp for 20 30 minutes we recommend rolling the vial vigorously between gloved hands to ensure it is fully thawed The dye is light sensitive Do not expose the Dye solution or Gel Dye to light for any length of time Keep the prepared Gel Dye solution in the dark Do not exceed 9300 rcf when filtering Gel Dye solution Exceeding 9300 rcf will change the properties of the gel 1 Vortex the thawed Dye Solution for 20 seconds before use 2 Transfer 27 uL of Protein Express Dye Solution blue cap to 1 vial of the Protein Express Gel Matrix red cap 3 Vortex and invert the tube several times until the solution is well mixed and spin it down for a few seconds Note Gel matrix is extremely viscous Make sure t
27. rom the date of shipment All claims under this warranty must be made within thirty days of the discovery of the defect Protein Express Assay User Guide LabChip Kit Essential Practices 26 Reverse Pipetting Technique Reagents PN CLS140156 Rev D Fv 4 STEP 1 STEP 2 STEP 3 STEP 4 Figure 16 Reverse pipetting Depress the pipette plunger to the first stop then continue to depress the plunger past the first stop but not all the way to the second stop Aspirate the selected volume plus an excess amount from the tube Leave the pipette tip in the solution for 5 10 seconds to ensure that the aspiration is complete Dispense the selected volume into the corner of the well by depressing plunger to the first stop Hold the pipette for 10 seconds with the plunger at the first stop to ensure that the dispense is complete Withdraw the pipette from the well Retain the excess volume in the pipette tip if performing an additional Reverse Pipetting transfer or return the excess volume to the source container for later use Store reagents at 4 C when not in use Protect the dye Gel Dye mixture and marker from light Store in dark and at 4 C when not in use The Gel Dye mixture expires 3 weeks after preparation For optimal performance use one reagent kit per chip The Protein Express Reagent Kit contains the reagents to run 384 samples equivalent to four chip preparations in the high throughput mode or eight chip prep
28. rs Salts and Additives Concentration Concentration Buffer amp Salts Limit Additives Limit Tris Chloride 250 mM Octyl Glucoside 2 5 Tris Glycine 250 mM Pluronic F68 0 1 HEPES 500 mM Sarcosyl 10 PBS 8 X CHAPS 0 5 Sodium Citrate 150 mM Tween 20 0 8 Sodium Phosphate 250 mM Triton X 100 0 6 Sodium Acetate 600 mM SDS 2 Sodium Chloride 1000 mM Zwittergent 3 14 0 4 Sodium Azide 6 PEG 3350 1 Sodium Hydroxide 500 mM Glycerol 30 Potassium Chloride 900 mM Urea 8M Ammonium 1000 mM Sucrose 1M Bicarbonate Magnesium 300 mM DMSO 25 Chloride Imidazole 900 mM EDTA 100 mM PhosphoSafe Ethanol 50 BugBuster 2 5 X BPER POP Culture Insect POP Culture Table 7 Incompatible Buffers Salts and Additives Buffer amp Salts Concentration Limit Additives Concentration Limit RIPA All PN CLS140156 Rev D Protein Express Assay User Guide Chip Well Aspiration Using a Vacuum 32 Chip Well Aspiration Using a Vacuum Aspirating with a pipette can leave used reagents in the chip wells For this reason PerkinElmer recommends vacuuming the wells instead This can be accomplished by attaching a permanent pipette tip to a house vacuum line with trap Figure 19 To avoid contamination use a new disposable pipette tip over the permanent tip for each chip aspirated Figure 20 Figure 19 A Permanent pipette tip attached
29. the active wells twice with water Milli Q or equivalent and store the chip with 120 uL of water in each active well e Thoroughly aspirate all fluid from the chip wells using a vacuum line e Each active well 1 2 3 4 7 8 9 and 10 should be rinsed and completely aspirated twice with water Milli Q or equivalent Do not allow active wells to remain dry e Add 120 uL of water Milli Q or equivalent to each active well 1 2 3 4 7 8 9 and 10 e Touch the Unload Chip button on the Home screen and place the chip into the instrument For GXII press the Chip button and load the chip into the instrument PN CLS140156 Rev D Protein Express Assay User Guide LabChip Kit Essential Practices 28 e Close the chip door securely For GXIl close the chip cartridge push the chip cartridge in and press the Eject button Transfer 750 uL of water Milli Q or equivalent into the Buffer Tube Install into the instrument Touch the Wash button on the Home screen Then touch the Wash button on the Wash screen Figure 17 R Chip Status Type HT Protein Express Chip Reagents N A Chip Expiration Jan 1 2020 Chip Life 400 Samples Left Status Buffer Figure 17 Wash screen After completion of the wash cycle open the chip cartridge and return the chip to the chip container ensuring the sipper is immersed in fluid e Thoroughly aspirate all fluid from the chip wells using a vacuu
30. ubble Rerun these sample wells Check the plate definitions Check the sample wells especially around the edge of the plate where evaporation is fastest and make a fresh plate if volumes are low If you suspect there may be debris in your samples spin the sample plate down in a centrifuge e g 3000 rcf for 5 minutes Unclog the sipper by repriming the chip See LabChip Kit Essential Practices on page 25 for instructions on how to reprime the chip Symptom No ladder peaks but sample peaks and marker peaks are present Possible causes 1 Low or no ladder volume in the Ladder Tube What to do 1 Add more ladder to the Ladder Tube and restart the run Recommended standard ladder volume is 120 uL minimum volume is 100 uL Symptom No marker peaks but sample peaks are present Possible causes 1 2 No marker added to chip well 4 If there is marker solution in chip well 4 the problem may be due to a marker channel clog What to do 1 PN CLS140156 Rev D This may be due to not filling the marker well or the chip remaining idle on the instrument for extended period of time Add or replenish the marker solution in the chip using the following procedure e Touch the Unload Chip button on the Home screen to open the chip door For GXII press the Chip button on the front of the instrument e Return the chip to the chip container ensuring the sipper is immersed in fluid e Thoroughly aspirate al
31. your conditions are not listed contact PerkinElmer see page 33 for more information on compatibility Particulates Sample plates should be spun down prior to analysis All buffers should be filtered with a 0 22 um cellulose acetate filter Salt Concentration Total salt concentration must not exceed 1M PN CLS140156 Rev D Protein Express Assay User Guide Specifications 4 Kit Contents Storage Prior to use store chips and reagents refrigerated at 4 C After use leave chips at room temperature for up to 30 days Table 3 Protein Express Reagent Kit Contents P N CLS960008 Reagent Vial Quantity Dye Solution Blue 1 vial 0 12 mL Sample Buffer White 8 vials 1 5 mL each Protein Gel Matrix Red 4 vials 0 7 mL each Protein Ladder Yellow 2 vials 0 08 mL each Lower Marker Green 2 vials 0 5 mL each Wash Buffer Purple 4 vials 1 8 mL each Protein Gel Matrix Orange 1 vial 1 3 mL Destain Table 4 Consumable Items Item Supplier and Catalog Number Quantity Spin Filters Costar Cat 8160 10 Ladder Tubes Genemate Cat C 3258 1 16 0 2 mL Detection Window VWR Cat 21912 046 1 Cleaning Cloth Swab ITW Texwipe Cat TX758B 3 Buffer Tubes E amp K Scientific Cat 697075 16 0 75 mL NC Table 5 Protein Express LabChips Item Part Number Protein Express LabChip for use with GXII 7
Download Pdf Manuals
Related Search