HT Protein Express LabChip Kit, Version 2 LabChip GXII User Guide
Contents
1. 6 Centrifuge the solutions in both spin filters at 9300 rcf for 5 minutes at RT Remove the filters in the tubes and store in the dark until the chip is to be prepared 7 The volumes of Gel Dye and Destain solutions prepared are the required amount for one chip prep If you are preparing additional solution for future use store the remaining solutions in the dark at 4 C and use within 3 weeks Preparing the Sample Denaturing Solution 1 Pipette 700 uL of HT Protein Express Sample Buffer white cap into a 2 0 mL centrifuge vial 2 If samples need to be reduced add 24 5 uL of BME or DTT or 3 75 uL of 100 mM TCEP 3 Vortex for 10 seconds This volume of sample buffer and reducing agent is sufficient to prepare 96 samples Caliper Life Sciences Page 5 of 21 Revised Nov 2010 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 522 2447 Tir 2 Y Caliper ou ifeSciences HT Protein Express LabChip Kit Version 2 LabChip GXII User Guide Preparing the Protein Samples and Ladder Note Samples can be prepared in either a 96 well or 384 well plate or in 0 6 mL microcentrifuge tubes and subsequently pipetted into a plate Procedures for both are described here 10 11 12 13 For each sample 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 microcentrifu2. 20 4 kDa 28 9 kDa 48 4 kDa 68 4 kDa and 119 2 kDa respectively 1 i Lage pee E e derl Ladder a T E a LL 14 1b Wee eae ce eco TING E Caliper Life Sciences Page 10 of 21 Revised Nov 2010 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 522 2447 T p Y Caliper ifeSciences HT Protein Express LabChip Kit Version 2 LabChip GXII User Guide Troubleshooting 1 Symptom Missing sample AND marker peaks Possible causes A Clog in sipper or marker channel of chip What to do A Reprime the chip See the section entitled LabChip Kit Essential Practices Chips for instructions on how to reprime the chip 2 Symptom Ladder detected but no sample peaks Possible causes A The sipper is not reaching the sample due to low sample volume in the well plate B Ifthe missing sample peaks occurred only in a few wells of the plate check those wells for air bubbles C The sipper is not reaching the sample due to an incorrect capillary height setting D Ifthe plate has been uncovered for some time sample evaporation might have occurred E Debris from the sample or sample prep is clogging the sipper What to do A Add more sample to the well Recommended sample volume is 44 uL B Manually insert a larger volume pipette tip 100 uL into the sample well and dislodge the bubble Rerun these sample wells C Re teach the robot positioning as described in LabChip GX Users Manua
3. Insert the Ladder Tube into ladder slot on the LabChip GX instrument Preparing the Buffer Tube 1 Add 750 uL of HT Protein Express Wash Buffer purple cap to the 0 75 mL Buffer Tube provided with the reagent kit Insert the Buffer Tube into the buffer slot on the LabChip GX instrument Note Replace the Buffer Tube with a freshly prepared Tube every 8 hours when the chip and instrument are in use Caliper Life Sciences Page 6 of 21 Revised Nov 2010 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 522 2447 2 Caliper Y TOA HT Protein Express LabChip Kit Version 2 LabChip GXII User Guide Preparing the Protein Chip 1 Allow the chip to come to room temperature and remove foil cover from chip wells 2 Use a pipette tip attached to a vacuum line to dry the top and bottom chip surfaces and the top of the chip container DO NOT run the tip over the central region of the detection window Return the chip to the chip container when the bottom of the chip and the top of the chip container are dry 3 Thoroughly aspirate all fluid from the chip wells using a vacuum line with the chip placed in the container and the sipper immersed in fluid Jaing a vacuumte aspirate the chip 4 Each active chip well 1 2 3 4 7 8 9 and 10 should wells is more effective than using a be rinsed and completely aspirated twice with molecular biology grade Pipette See page 20 for more details water Do not allow active wells
4. 2 LabChip GXII User Guide Safety Warnings and Precautions WARNING 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 Dye Solution contains DMSO Avoid contact with skin and eyes WARNING Dye Solution contains SDS Avoid inhalation and contact with skin and eyes WARNING Wash Buffer and Sample Buffer contain LDS Avoid inhalation and contact with skin and eyes WARNING Gel Matrix contains Methylurea Avoid contact with skin and eyes Storage Prior to use store chips and reagents refrigerated at 4 C After use leave chips at room temperature for up to 30 days Caliper Life Sciences Page 4 of 21 Revised Nov 2010 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 522 2447 T p Y Caliper ifeSciences HT Protein Express LabChip Kit Version 2 LabChip GXII User Gui
5. LabChip Kit Version 2 LabChip GXII User Guide Reordering Information Product Part Number HT Protein Express LabChip Kit Version 2 760499 HT Protein Express Reagent Kit 760328 Buffer Tube E amp K Scientific 697075 NC Ladder Tube Genemate C 3258 1 2 0 ml Centrifuge Tubes Eppendorf 022363352 Customer Technical Support Caliper Life Sciences 68 Elm Street Hopkinton MA 01748 1668 Phone 1 877 LABCHIP 522 2447 Fax 1 508 435 3439 For additional assay and instrument troubleshooting refer to the LabChip HT Software Help file Call Caliper Technical Support at 1 877 LABCHIP 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 User Guide Caliper the Caliper logo LabChip and the LabChip logo are registered trademarks of Caliper Life Sciences The reagent kit contains materials manufactured for Caliper by Molecular Probes Inc and are provided under a license from Molecular Probes Inc for research use only Copyright Caliper Life Sciences 2010 http www caliperLS com Doc 450589 Caliper Life Sciences Page 21 of 21 Rev
6. ee x Caliper Y TOA HT Protein Express LabChip Kit Version 2 LabChip GXII User Guide Inserting a Chip into the LabChip GX Instrument _ 1 Check that the sample plate Buffer Tube J sample Well At and Ladder Tube are placed appropriately on the instrument 2 Remove the chip from the chip storage container and inspect the chip window Clean BOTH sides of the chip window with the Caliper supplied cleanroom cloth dampened with a 70 isopropanol solution in DI water Eject the chip cartridge by pressing the CHIP button on the instrument front panel Release the cartridge latch insert the chip into the LabChip GX instrument refasten the latch and push the cartridge into the instrument 5 Press the EJECT button on the instrument front panel to retract the sample plate and send the sipper to the Buffer Tube Running the HT Protein Express Assay Start the LabChip GX software On the main screen click on the Run button in the upper left corner of the LabChip GX Software The Start Run dialog box will pop up with tabs listed as Output Run and Advanced gt w LS In the Run Tab select the appropriate assay type operator name plate name well pattern and barcode option e For HT Protein Express assays appropriate assay types are o HT Protein Express 100 For sizing of protein in 14kDa to 100 kDa range o HT Protein Express 100 High Sensitivity For sizing of protein in 14kDa to 100 kDa range Greater sensitivity but req
7. help avoid introducing bubbles into the chip when pipetting the gel v a a i Reverse Pipetting Technique Step 1 Depress the pipette plunger to the second stop Step 2 Aspirate the selected volume plus an excess amount from the tube Step 3 Dispense the selected volume into the corner of the well by depressing plunger to the first stop Step 4 Withdraw the pipette from the well STEP 1 STEP 2 STEP 3 STEP 4 Caliper Life Sciences 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 from the date of shipment All claims under this warranty must be made within thirty days of the discovery of the defect Caliper Life Sciences Page 16 of 21 Revised Nov 2010 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 522 2447 T p Y Caliper ifeSciences HT Protein Express LabChip Kit Version 2 LabChip GXII User Guide Reagents e Store reagents at 4 C when not in use e Protect the dye Gel Dye mixture and marker from light Store in dark and at 4 C when not in use e The Gel Dye mixture expires three weeks after preparation e For optimal performance use one reagent kit per chip The HT Protein Express Reagent Kit contains the reagents to run three 96 well plates or three chip preparations whichever comes first e Repriming Chips Press the CHIP button on the front instrument panel to e
8. to remain dry 5 Add 75 uL of Gel Dye solution to chip wells 3 7 8 and 120 uL to well 10 using a Reverse Pipetting Technique Reagent Placement 6 Add 75 uL of Destain solution to chip wells 2 and 9 using a Reverse Pipetting Technique 7 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 HT 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 40 L Make sure the marker volume is pipetted accurately If there is not enough marker in chip well 4 the marker will deplete prior to the completion of the plate Data collected without marker peaks cannot be analyzed by the software iii T P Destain el Dye Make sure the rims of the chip wells are free of adhesive residue Place the chip in the LabChip GX instrument to begin the assay Add Marker Gel Dye and Destain according to the image Note Be sure to periodically clean the O rings on the top plate above of the chip interface on the LabChip GX instrument Use the provided lint free swab dampened with DI water or 70 isopropanol solution in DI water to clean the O rings using a circular motion Allow the O rings to dry before inserting a chip Caliper Life Sciences Page 7 of 21 Revised Nov 2010 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 522 2447
9. with specific buffers salts and additives If your conditions are not listed please contact Caliper Life Sciences at 1 877 LABCHIP 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 Sait Concentration Total salt concentration must not exceed 1M 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 Expected chip lifetime is based on use under normal laboratory conditions and adherence to Caliper preparation protocols sample guidelines and storage conditions Individual laboratory results may vary Caliper Life Sciences Page 2 of 21 Revised Nov 2010 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 522 2447 ay A 7 YW Caliper ifeSciences HT Protein Express LabChip Kit Version 2 LabChip GXII User Guide HT Protein Express Reagent Kit Part Number 760328 wey poenom e O foom C Sn HT Protein Express LabChip Kit Version 2 Part Number 760431 eww oo p a menee E eono E a aon s oo o e Buffer Tubes p10 0 75 mL tubes HT Protein Express Reagent Kit Caliper Life Sciences Page 3 of 21 Revised Nov 2010 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 522 2447 T p Y Caliper ifeSciences HT Protein Express LabChip Kit Version
10. 8 and 10 using a Reverse Pipetting Technique e Add 75 L of Destain solution to chip wells 2 and 9 using a Reverse Pipetting Technique e 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 HT Protein Express Marker green cap to chip well 4 If the chip will only Caliper Life Sciences Page 17 of 21 Revised Nov 2010 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 522 2447 Tir 2 Y Caliper eu ifeSciences HT Protein Express LabChip Kit Version 2 Samples Caliper Life Sciences 68 Elm Street LabChip GXII User Guide 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 40 uL Place the chip in the LabChip GX instrument Reinsert the cartridge by engaging the latch and pushing the cartridge back into the instrument Press the Run button on the main screen of the LabChip GX software Other Considerations New protein chips should be stored refrigerated prior to first use After running the first set of samples protein 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 should not be exposed to an open environment for lo
11. CHIP 522 2447 T p Y Caliper ifeSciences HT Protein Express LabChip Kit Version 2 LabChip GXII User Guide LabChip Kit Essential Practices To ensure proper assay performance please follow the important handling practices described below Failure to observe these guidelines 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 LabChip HT Software Help file or call Caliper Technical Support at 1 877 LABCHIP General e Allow the chip sample plate and all reagents to equilibrate to room temperature before use approximately 20 to 30 minutes e 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 e 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 e Calibrate laboratory pipettes regularly to ensure proper reagent dispensing e Only the Caliper supplied clean room cloth can be used on the chip to clean the detection window e Water used for chip preparation procedures must be 0 22 micron filtered deionized molecular biology grade e Use of the Reverse Pipetting Technique described below will
12. de Preparation Procedures Additional Items Required e 0 6 mL centrifuge tubes and or 96 well plates for denaturing protein samples Note Avoid using non stick varieties of lab consumables These may induce unexpected or erratic assay results caused by surface treatments leaching into dye or gel components e MilliQ Water Molecular biology grade or better 0 22 micron filtered e 70 isopropanol solution in DI water e Reducing agents BME beta mercaptoethanol 1M DTT dithiothreitol or 100mM TCEP Note Allow the chip and all reagents to equilibrate to room temperature before use aoproximatelv 20 to 30 minutes Preparing the Gel Dye and Gel Destaining Solutions Note The Dye Solution contains DMSO and must be thawed completely before use Note Do not exceed 9300 rcf when filtering gel and Gel Dye solutions Exceeding 9300 rcf will change the properties of the gel 1 Gently vortex the thawed Dye Solution for 10 seconds before transferring 2 Transfer 20 uL of HT Protein Express Dye Solution blue cap to the 2 0 mL centrifuge tube provided with the reagent kit Add 520 uL of the HT Protein Express Gel Matrix red cap using a Reverse Pipetting Technique Vortex the solution until it is well mixed Transfer the solution into one of the spin filters provided with the reagent kit 5 Transfer 250 uL of the HT Protein Express Gel Matrix red cap into a separate spin filter This will be used as the Destain solution
13. e or use a direct displacement pipette Prepare a fresh Gel Dye solution Wash and reprime the chip with the new Gel Dye mixture See the section entitled LabChip Kit Essential Practices Chips for instructions on how to wash and reprime the chip 7 Symptom No marker peaks but sample peaks are present Possible causes A No marker added to chip well 4 B lf there is marker solution in chip well 4 the problem may be due to a marker channel clog What to do A This may be due to not filling marker well or chip remaining idle on instrument for extended period of time Add or replenish the marker solution in the chip using the following procedure Press the CHIP button on the front instrument panel to eject the chip cartridge e 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 chip well 4 using a vacuum line e Ensure that chip well 4 is rinsed and completely aspirated twice with molecular biology grade water Caliper Life Sciences Page 13 of 21 Revised Nov 2010 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 522 2447 T p Y Caliper ifeSciences HT Protein Express LabChip Kit Version 2 LabChip GXII User Guide e Add Protein Express Dye Solution to chip well 4 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 HT Protein Express Marker green cap to chi
14. ge 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 HT Protein Express Ladder to warm up to room temperature for 15 20 minutes followed by vortexing for 10 seconds Pipette 12 uL of HT Protein Express Ladder into a microcentrifuge tube Do not add denaturing solution to the ladder Denature samples and ladder at 100 C for 5 minutes Optimum denaturing conditions may vary by sample type Tap or spin the sample plate to move the fluid to the bottom of the wells Spin the HT Protein Express Ladder and sample tubes if used for 15 seconds using a mini centrifuge Add 120 uL of water to the HT Protein Express Ladder Vortex the ladder mixture for a few seconds to achieve good mixing Add 35 uL of water to each sample well or sample tube This step should not be done more than an hour before starting the assay If running High Sensitivity scripts add 32 uL of water to each 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 If the samples are prepared in tubes load 44 uL of each sample onto a 96 well plate Spin the sample plate to eliminate bubbles and move the fluid to the bottom of the wells Place the sample plate onto the instrument s plate holder Transfer 120 uL prepared ladder to the provided 0 2 mL Ladder Tube
15. gration is observed repeatedly on a new chip then return the chip to Caliper Life Sciences along with the data file for replacement Caliper Life Sciences Page 14 of 21 Revised Nov 2010 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 522 2447 T p Y Caliper ifeSciences HT Protein Express LabChip Kit Version 2 LabChip GXII User Guide 9 Symptom Peaks migrating much later than expected Possible causes A Particulates from the samples may be clogging the separation channel B Excess dye within the separation channel C Gel Dye was not primed properly into the chip What to do A Minimize the loading of particulates in the sample by performing a centrifuge spin of the sample plate e g 3000 rcf for 5 mins and or ensuring the Sip Middle plate type is selected in the Start Run dialog box before starting a new run The debris maybe flushed out of the chip by washing and re priming the chip See the section entitled LabChip Kit Essential Practices Chips for instructions on how to wash and reprime the chip B Prepare a fresh Gel Dye solution Wash and reprime the chip with the new Gel Dye mixture See the section entitled LabChip Kit Essential Practices Chips for instructions on how to wash and reprime the chip C Check the O rings on the top surface of the chip interface and clean if necessary Caliper Life Sciences Page 15 of 21 Revised Nov 2010 68 Elm Street Hopkinton MA 01748 1668 1 877 LAB
16. ised Nov 2010 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 522 2447
17. ithin 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 B Use the provided lint free swab dampened with DI water or 70 isopropanol solution in DI water to clean 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 A To reduce pressure leaks at the chip interface clean the O rings frequently Remove the O rings from the top plate of the chip interface on the LabChip GX instrument Soak O rings in DI water for a few minutes Clean the O ring faces by rubbing between two fingers B 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 DI water C Allow the O rings and chip interface to air dry Reinsert the O rings into the chip cartridge Caliper Life Sciences Page 9 of 21 Revised Nov 2010 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 522 2447 y Caliper ifeSciences HT Protein Express LabChip Kit Version 2 LabChip GXII User Guide Results HT Protein Express Ladder Result The electropherogram of a typical Protein Express ladder is shown below 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
18. ject the chip cartridge Reinsert the cartridge by pushing the cartridge back into the instrument Press the Run button on the main screen of the LabChip GX software e Washing and Repriming Chips Press the CHIP button on the front instrument panel to eject the chip cartridge e 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 vacuum line e Each active well 1 2 3 4 7 8 9 and 10 should be rinsed and completely aspirated twice with molecular biology grade water Do not allow active wells to remain dry e Add 75 uL of Wash Buffer to each active well 1 2 3 4 7 8 9 and 10 e Place the chip in the LabChip GX instrument e Reinsert the cartridge by engaging the latch and pushing the cartridge back into the instrument e Press the Wash button on the main screen of the LabChip GX software After completion of the wash cycle press the CHIP button on the front instrument panel to eject the chip cartridge e 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 vacuum line e Each active well 1 2 3 4 7 8 9 and 10 should be rinsed and completely aspirated twice with molecular biology grade water Do not allow active wells to remain dry e Add 75 uL of Gel Dye solution to chip wells 3 7
19. l D Check the sample wells especially around the edge of the plate where evaporation is fastest and make a fresh plate if volumes are low E If you suspect there may be debris in your samples spin the sample plate down in a centrifuge Unclog the sipper by repriming the chip See the section entitled LabChip Kit Essential Practices Chips for instructions on how to reprime the chip Caliper Life Sciences Page 11 of 21 Revised Nov 2010 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 522 2447 T p Y Caliper ifeSciences HT Protein Express LabChip Kit Version 2 LabChip GXII User Guide 3 Symptom No ladder peaks but sample peaks and marker peaks are present Possible causes A Low or no ladder volume in Ladder Tube What to do A Add more ladder to the Ladder Tube and restart the run Recommended standard ladder volume is 120 uL minimum volume no less than 100 uL 4 Symptom Hump in several electropherograms which do not correspond to sample data 1250 1000 o 750 O Cc D O D 5 900 m L 250 ae _ f J J a NG J 0 aot er tg py l 00 D E 20 25 30 35 40 45 50 55 60 Time seconds Possible causes A Electrode 7 is dirty and has contaminated the Gel Dye mixture in well 7 B High concentrations of detergent in the sample buffer can sometimes cause humps in the electropherogram at lower molecular weights approxi
20. m Azide Sodium Hydroxide Potassium Chloride Ammonium Bicarbonate Magnesium Chloride Imidazole PhosphoSafe BugBuster BPER POP Culture Insect POP Culture 250 mM 250 mM 500 mM 8 X 150 mM 250 mM 600 mM 1000 mM 6 500 mM 900 mM 1000 mM 300 mM 900 mM Octyl Glucoside Pluronic F68 Sarcosyl CHAPS Tween 20 Triton X 100 SDS Zwittergent 3 14 PEG 3350 Glycerol Urea Sucrose DMSO EDTA Ethanol Incompatible Buffers Salts and Additives Buffer amp Salts RIPA Caliper Life Sciences 68 Elm Street Concentration Limit All Hopkinton MA 01748 1668 1 877 LABCHIP 522 2447 Additives Page 19 of 21 2 5 0 1 10 0 5 0 8 0 6 2 0 4 1 30 8M 1M 25 100 mM 50 Concentration Limit Revised Nov 2010 Tr x Caliper Y TOA HT Protein Express LabChip Kit Version 2 LabChip GXII User Guide Chip Well Aspiration Using a Vacuum Aspirating with a pipette can leave used reagents in the chip wells For this reason Caliper recommends vacuuming the wells instead This can be accomplished by attaching a permanent pipette tip to a house vacuum line with trap Figures 1a and b To avoid contamination use a new pipette tip over the permanent tip for each chip aspirated Figure 2 Figure 1a Figure 1b Figure 2 Caliper Life Sciences Page 20 of 21 Revised Nov 2010 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 522 2447 T p Y Caliper ifeSciences HT Protein Express
21. mately 6 20 kDa What to do A Remove the chip and follow the electrode cleaning procedure We also recommend using the provided swab and isopropanol to manually clean electrode 7 Before restarting the run lower the detergent concentration in the sample see table for compatibility of buffers and detergents Caliper Life Sciences Page 12 of 21 Revised Nov 2010 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 522 2447 T p Y Caliper ifeSciences HT Protein Express LabChip Kit Version 2 LabChip GXII User Guide 5 Symptom High baseline fluorescence for example greater then 1000 counts Possible causes A The destain wells 2 and 9 do not contain Destain solution gel matrix with no dye B 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 A Prepare a fresh Destain solution Wash and reprime the chip with the new Destain solution See the section entitled LabChip Kit Essential Practices Chips for instructions on how to wash and reprime the chip B See A 6 Symptom Lower signal for ladders and samples than expected Possible causes A Improper SDS concentration in Gel Dye matrix What to do A Ensure that Dye Concentrate is completely thawed and mixed Ensure proper dispense of viscous gel into the Gel Dye mixture e g use a Reverse Pipetting Techniqu
22. ng 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 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 Caliper 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 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 Page 18 of 21 Revised Nov 2010 Hopkinton MA 01748 1668 1 877 LABCHIP 522 2447 T p Y Caliper ifeSciences HT Protein Express LabChip Kit Version 2 LabChip GXII User Guide Protein Assay Buffer Salt and Additive Compatibility Compatible Buffers Salts and Additives Concentration Additives Limit Concentration Buffer amp Salts Limit Tris Chloride Tris Glycine Hepes PBS Sodium Citrate Sodium Phosphate Sodium Acetate Sodium Chloride Sodiu
23. p 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 40 uL e Reinsert the cartridge by engaging the latch and pushing the cartridge back into the instrument e Press the Run button on the main screen of the LabChip GX software e Perform a marker channel unclogging procedure by repriming the chip See the section entitled LabChip Kit Essential Practices Chips for instructions on how to reprime the chip 8 Symptom Peaks migrating much faster than expected NOTE Some migration time variance between chips or within a plate is considered normal chip performance All chips are QC tested at Caliper Life Sciences prior to shipment and normal migration time windows for the lower markers are listed below HT Protein Express Assay Lower Marker 11 to 13 5 seconds Upper Ladder Protein 23 32 seconds on first plate Upper Ladder Protein 20 29 seconds on third plate Possible causes A Incorrect Gel Dye concentration 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 What to do A Prepare a fresh Gel Dye solution Wash and reprime the chip with the new Gel Dye mixture See the section entitled LabChip Kit Essential Practices Chips for instructions on how to wash and reprime the chip B If fast mi
24. uires a larger amount of sample Slightly lower resolution may be observed o HT Protein Express 200 For sizing of protein in 14kDa to 200 kDa range o HT Protein Express 200 High Sensitivity For sizing of protein in 14kDa to 200 kDa range Greater sensitivity but requires a larger amount of sample Slightly lower resolution may be observed 5 Inthe Outout Tab select the destination of the file the filename convention and any additional data to autoexport 6 Inthe Advanced Tab select the number of times each well is sampled the inclusion of any sample names and any expected peaks 7 Click Start to begin the run Caliper Life Sciences Page 8 of 21 Revised Nov 2010 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 522 2447 T p Y Caliper ifeSciences HT Protein Express LabChip Kit Version 2 LabChip GXII User Guide Storing the Protein Chip After use the chip must be cleaned and stored in the chip container 1 Remove the reagents from each well of the chip using vacuum 2 Each active well 1 2 3 4 7 8 9 and 10 should be rinsed and aspirated twice molecular biology grade water Add 120 uL molecular biology grade water to the active wells Place the chip in the plastic storage container The sipper should be submerged in the fluid reservoir Cover the wells with parafilm to prevent buffer evaporation and store the chip at room temperature until next use The chip must be used to its lifetime three 96 well plates w
25. yy Cali er ifeSciences HT Protein Express LabChip Kit Version 2 LabChip GXII User Guide Contents Specifications Sample Conditions Kit Contents Preparation Procedure Additional Items Required Preparing the Gel Dye and Gel Destaining Solutions Preparing the Sample Denaturing Solutions Preparing the Protein Samples and Ladder Preparing the Buffer Tube Preparing the Protein Chip Inserting a Chip into the LabChip GX Instrument Running the HT Protein Express Assay Storing the Protein Chip Chip Cartridge Cleaning Typical Results Troubleshooting LabChip Kit Essential Practices General Reagents Chips Samples Protein Assay Buffer Salt and Additive Compatibility Chip Well Aspiration Using a Vacuum Reordering Information Customer Technical Support Caliper Life Sciences Page 1 of 21 68 Elm Street Hopkinton MA 01748 1668 1 877 LABCHIP 522 2447 OO 0O NOO O 01 OI QI N N oh 11 Revised Nov 2010 T p Y Caliper ifeSciences HT Protein Express LabChip Kit Version 2 LabChip GXII User Guide Specifications Sizind Rande P100 Assay 14 kDa 100 kDa g nang P200 Assay 14 kDa 200 kDa Sizing Resolution 10 difference in molecular weight 30 CV up to 120 kDa Above 120 kDa quantitation is not specified Sample Conditions Buffers Salts and Please refer to the list Protein Assay Buffer Salt and Additive Compatibility in Additives the rear of the guide for compatibility
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