0791 v1.0 Proseek User Manual - final
Contents
1. Fig 3 Fig 4 Proseek Assay Development kit 5 5 Add the Pre Extension Solution to dilute the Proseek probes and lower their effective concentration Subsequently add the Extension master mix which will extend oligos of two Proseek probes that are bound to a target protein through a DNA polymerization event creating the real time PCR amplicon Fig 5 6 Combine the Real Time PCR master mix with the extension products 8 Fig 6 7 Amplify the DNA using your standard real time PCR instrument Fig 7 8 Analyze your real time PCR data in a spreadsheet software e g Microsoft Excel Ct Concentration Fig 8 6 Proseek Assay Development kit 3 Application 3 1 DETECT AND QUANTIFY PROTEIN EXPRESSION Proseek is intended to be used for detection and quantification of a single protein in a solution e g plasma and serum 1 ul of sample is needed per reaction and the target protein is detected using one single antigen affinity purified polyclonal antibody batch or two matched monoclonal antibodies uy N Fig 9 Single protein detection Proseek Assay Development kit 7 8 4 Reagents and equipment 4 1 PROSEEK ASSAY DEVELOPMENT KIT REAGENTS When setting up a new Proseek assay you need the following Proseek Assay Development kit components b Proseek Probemaker A to create Pros2. reagents technique during reagent and sample transfer steps Avoid bubble formation Partial evaporation of Ensure that the adhesive plastic film or tube lids is samples completely sealed during the Probe incubation Extension and Real Time PCR Contamination from Be careful when removing the adhesive plastic film or tube other wells Real time PCR instrument is not operating well lids after the Probe incubation and the Extension to avoid contam or lids pipette Run rea ips ination drops from other wells Before removing the film centrifuge the plate briefly Also remember to change between sample and reagent transfer steps time PCR replicates of any real time PCR master mix not necessarily Proseek reagents CV should be below 8 on a linear scale if not recalibrate the instrument and contact the manufacturer If problems remain please contact us at support olink com or 46 18 444 3970 Proseek Assay Development kit 23 Appendix 1 96 well plate template Proseek Assay Development 25 26 Proseek Assay Development kit This product is for research use only Not for use in human diagnostic or therapeutic procedures This product includes a license for non commercial use of the Proseek product Commercial users will require additional licenses Please contact Olink AB for details There are no warranties expressed or implied which extend beyond this description O
3. Proseek Assay Development kit OLINK BIOSCIENCE The protocols in this manual are compatible with Proseek Probemaker Art no 93001 0010 Proseek Probemaker B Art no 93002 0010 and Proseek Assay Reagents in reactions size 100 and 1000 Art no 93003 0100 and Art no 93003 1000 Table of content T INTRODUCTION hee E 4 2 PRINCIPLE OF THE ASSAY notet oerte rem pte emis 5 3 APPLICATION 7 3 1 Detect and quantify protein expression 7 4 REAGENTS AND EQUIPMENT 8 41 Proseek Assay Development kit reagents 8 42 Reagents to be supplied by the user 9 43 Equipment needed 9 44 Software for analysis s 9 5 ASSAY CONSIDERATIONS 10 5 1 Antibodies 10 5 2 Sample material 10 5 3 Validation of assay 11 6 REAGENT HANDLING AND STORAGE 1 st 12 6 1 Proseek Probemaker 6 2 Proseek Assay Reagents 72 PROTOCOLS r nda weso d NE tender tpe t QIRD NADIN 14 7 1 Proseek Probemaker protocol 14 7 2 Proseek Assay protocol 15 B RESULTS yane ne are KAN dnte tnt 19 8 1 Typical results 19 82 Analysis of real time PCR data 20 9 TIPS AND TRICKS 21 9 1 Precision 21 92 Sensitivity 21 9 3 Quantification 21 10 TROUBLESHOOTIN
4. Unknown 33 83272 Unknown 33 811447 Unknown 33 326935 dne mun o Media Quy Quem Quy CS UC EW Posed Ad FAM itii E 1 CtRep 2 Avg Ct Stdev Ct 0 33 9 33 7 33 8 0 09 0 01 33 3 32 7 33 0 0 42 0 1 32 0 32 0 32 0 0 00 1 30 9 30 6 30 7 0 21 10 29 0 29 1 29 0 0 04 100 27 1 26 7 26 9 0 26 1000 25 7 25 1 25 4 0 44 10000 27 2 26 6 26 9 0 41 Fig 10 Typical result file from the real time PCR instrument Applied Biosystems 7900 Fig 11 Result file exported to Microsoft Excel Fig 12 Ct values in duplicates Rep 1 and Rep 2 average Ct Avg Ct and standard deviation Stdev Ct of each data point in the calibration curve Proseek Assay Development kit 19 255 26 4 27 4 m 28 4 29 amp 304 2 314 Fig 13 Calibration curve of VEGF R amp D 32 Systems Ab AF 293 NA and Ag 293 VE 010 0 10 000 pM with concentrations on the xaxis 334 and the average Ct values in reverse order 34 4 of duplicates on the y axis Error bars indicate 35 i x standard deviation 0 001 01 1 10 100 1000 10000 Concentration VEGF pM 8 2 ANALYSIS OF REAL TIME PCR DATA 8 2 1 ABSOLUTE QUANTIFICATION WITH LINEAR REGRESSION Calculate the concentrations in the calibration curve to log scale in a spreadsheet program e g Microsoft Excel exclude the zero buffer and concentrations that are not within the linear ran
5. approximately 1200 reactions If your total number of reactions in one experiment is 40 or more we recommend you to prepare master mixes for 8 additional reactions Before starting 1 Please read through the entire Proseek Assay protocol 2 Decide how many samples you will include in the experiment and the number of replicates 3 Plan how to prepare the calibration curve number of concentrations dilution factor and number of replicates 4 Calculate the total number of reactions in the experiment 5 Use the 96 well plate template in Appendix A on page 24 and select a location for each reaction 1 DILUTION OF PROSEEK PROBES FOR 3 MONTH STORAGE a Thaw the Probe Diluent vortex and quick spin b Dilute Proseek probes A and from Probemaker Storage Solution 130 nM 1 200 in the Probe Diluent in separate tubes add 1 ul of each Proseek probe to 199 yl of Probe Diluent The final concentration is now 650 pM the Proseek probes can be stored in the Probe Diluent for 3 months at 4 2 PREPARATION OF CALIBRATION CURVE AND SAMPLES a Thaw the Calibrator Diluent and the Assay Solution vortex and quick spin b Prepare a serial dilution of your antigen standard in the Calibrator Diluent We recommend 7 concentrations in dilution steps of 1 10 in the calibration curve e g 10 nM 1 nM 100 pM 10 pM 1 pM 0 1 pM and 0 01 pM and a zero standard 0 pM 1 pM of a 10 kDa antigen corresponds to 10 pg ml After each
6. matrix effects are present in the sample Proseek Assay Development kit 11 12 6 Reagent handling and storage 6 1 PROSEEK PROBEMAKER Store the unused Proseek Probemaker kit at 20 C Once you have conjugated your antibody with Proseek Probemaker we recommend storage at 4 C in Storage Solution A or B The Storage Solution A and B contains buffer and reagents for stabilizing and preserving Proseek probes A and B Each Proseek Probemaker kit A or B contains reagents to conjugate 10 ug antibody at a concentration of 1 mg ml Proseek probes from one conjugation is sufficient for approximately 100 000 reactions Oligo A or B store at 20 C gt One vial with lyophilized activated oligonucleotide for one conjugation of 10 ug antibody at a concentration of 1 mg ml Conjugation Buffer store at 20 C b Thaw vortex and quick spin before use Ready to use solution Stop Reagent store at 20 C b Thaw vortex and quick spin before use b Ready to use solution Storage Solution A or B store at 20 C b Thaw vortex and quick spin before use b Ready to use solution Proseek Assay Development kit 6 2 PROSEEK ASSAY REAGENTS Store the unused Proseek Assay Reagents at 20 C Some solutions are supplied as 10x concentrated stocks Dilute required volumes of the stocks immediately before use Note Do not store diluted reagents Calibrator Diluent store at 20 C Thaw vortex and quick spin befo
7. CR master mix is thoroughly vortexed before addition to the samples Proseek Assay Development kit 13 7 Protocols 7 1 PROSEEK PROBEMAKER PROTOCOL The following Proseek Probemaker protocol describes how you conjugate either the Oligo A or the Oligo B to one antibody to create a Proseek probe A or B You can create the Proseek probes A and B in parallel however it is highly important to change pipette tips and gloves between the A and B conjugation in order to avoid cross contamination between the probes If a single polyclonal antibody batch is used split the antibody into two separate tubes with 10 ul in each and label one with Oligo A and the other with Oligo B Before starting 1 Please read through the entire Proseek Probemaker protocol 2 Ensure that your antibody or antibodies to be conjugated meet the requirements specified in Section 5 1 CONJUGATION OF PROSEEK PROBEMAKER OLIGO A OR B TO ANTIBODY 1 Add 1 ul of Conjugation Buffer to 10 ul of the antibody to be conjugated 2 Mix gently with a pipette 3 Transfer the antibody solution to one vial of lyophilized Oligo A or B 4 Mix gently with a pipette and make sure the oligo is completely dissolved 5 Incubate for 3 at 37 C 6 Add 1 ul of Stop Reagent to the reaction 7 Agitate gently 8 Incubate for 30 min at room temperature 9 Transfer 9 6 ul of the reaction to the vial with Storage Solution A or B S
8. G tte mrt tete 22 APPENDIX 1 96 WELL PLATE TEMPLATE s 24 Proseek Assay Development kit 3 4 1 Introduction Proseek reagents from Olink Bioscience enable detection and quantification of proteins in a solution such as plasma and serum ina minimal sample volume With the Proseek Assay Development kit a new assay for any target protein with appropriate available antibodies can rapidly be developed Combining the target specific antibodies with the Proseek reagents the protein of interest is detected using standard real time PCR The Proseek reagents are based on PEA which is a Proximity Extension Assay technology A pair of oligonucleotide labeled antibodies Proseek probes are allowed to pairwise bind to the target protein present in the sample in a homogeneous assay without washing steps When the two Proseek probes are in close proximity a new PCR target sequence is formed by a proximity dependent DNA polymerization event The resulting sequence is subsequently detected and quantified using real time PCR Proseek Assay Development kit 2 Principle of the assay 1 Create Proseek probes A and by conjugating your antibodies to Oligo A and B using Proseek Probemaker A and B 2 Make a dilution series of your antigen standard in Calibrator Diluent 3 Incubate dilution series and samples with Proseek probes A and 4 During the incubation Proseek probes will bind to the target protein
9. aster mix However this will give you a background level at a higher Ct value thus increasing the risk for lower precision Performing the probe incubation over night at 4 C instead of 2 h at room temperature may also help increasing the sensitivity 9 3 QUANTIFICATION To enhance the precision further of the quantification you can narrow the concentration range of the calibration curve closer to the protein levels of your samples and use a smaller dilution factor of the antigen standard e g 1 2 or 1 3 Proseek Assay Development kit 21 10 Troubleshooting Some general guidelines are given below PROBLEM PROBABLE CAUSE SUGGESTED SOLUTION Poor No or inefficient Run a polyacrylamide or agarose gel elecrophoresis with your standard conjugation with Proseek probes and free antibody to verify a conjugation with curve Proseek Probemaker Proseek Probemaker Contact support olink com to receive protocols and gel images of expected results No or insufficient Check the quality of the antigen Also ensure that your binding of Proseek antibody used for Probemaker conjugation has been raised probes to antigen against the selected antigen preferably the antigen should be purchased from the same supplier as the antibody Improper dilution of Ensure that the dilution series of the antigen standard has antigen standard been properly prepared No or inefficient Ensure that the Extension Polymerase has been stored at e
10. bation with samples and diluted antigen standard gt Pre Extension Solution 10x For dilution of the incubation reaction b Extension Solution 10x Contains all the components needed for the extension of the Proseek probe oligos except for the Extension Polymerase gt Extension Polymerase 5 U l b Real Time PCR Solution ready to use Contains all the components needed for real time PCR detection and quantification except for the Real Time PCR Polymerase Real Time PCR Polymerase 1 U l Proseek Assay Development kit 4 2 REAGENTS TO BE SUPPLIED THE USER One single antigen affinity purified polyclonal antibody batch or two matched monoclonal antibodies Antigen standard preferably from the same supplier as for the antibody recommended High purity water distilled MilliQ or similar 4 3 EQUIPMENT NEEDED v Yv Y Yv vvv vv wo wow v Microfuge tubes 96 well microplate or test tubes typically PCR tubes Optical 96 or 384 well microplate or test tubes for real time PCR detection Pipettes covering the range from 1 ul to 1000 yl Multi channel pipettes recommended Pipette tips Freezing block 20 for enzymes Thermal cycler with heated lid Incubator or oven 37 Vortexer Centrifuge for plates or tubes Adhesive plastic film Optical adhesive plastic film for real time PCR detection Real time PCR instrument Olink has tested Applied Biosystems 7900 Applied Biosystem
11. briefly Place the plate in your real time PCR instrument Thermal cycling conditions FAM as reporter and ROX as passive reference Daboyl is the quencher you may denote none or non fluorescent with equal performance gt 95 5 min b 95 15s 45 cycles 60 1 min Start the run When the run is finished remove and dispose of the plate 6 ANALYSIS Export the result file from the real time PCR instrument to a spreadsheet program e g Microsoft Excel Calculate the average Ct value and standard deviation of replicates for samples and data points in the calibration curve Plot the calibration curve with the concentrations on the and the average Ct values in reverse order on the y axis 18 Proseek Assay Development kit 8 Results 8 1 TYPICAL RESULTS 505 2 3 Absolute QuantificationResults 1 0 Filename PEA 076 110201 PlatelD Assay Absolute Quantification Run DateTime 2 1 11 2 30 58 PM Operator ThermalCycleParams Sample Information Position Flag Sample Detector Task AL Passed AL FAM Unknown 34 11196 A2 Passed A2 FAM Unknown 34 106636 A3 Passed FAM Unknown 33 55748 A4 Passed 4 FAM Unknown 33 853817 A5 Passed AS FAM Unknown 33 866272 Passed Unknown 33 74429 A7 Passed A7 FAM Unknown 33 94533 A8 Passed A8 FAM Unknown 34 02659 A9 Passed A9 FAM Unknown 33 60669 10 Passed 10 11 Passed All FAM 12 Passed 12
12. dilution mix gently by pipetting and then change to a new pipette tip c If necessary dilute your samples in the Calibrator Diluent Proseek Assay Development kit 15 3 PROBE INCUBATION a b 9 Prepare a probe master mix mixing Proseek probes from step 1b with the Assay Solution according to the table below and vortex briefly 3 of probe master mix is needed per reaction prepare an excess volume to account for pipetting losses 1 reaction ul reactions ul Proseek probe A 650 pM Proseek probe 650 pM Assay Solution 24 BM MM Total volume of probe master mix 2 Transfer ul of the probe master mix to each well of a 96 well plate or multi tube strip It is advisable to transfer the probe master mix to an 8 or 12 tube strip and use a 1 10 ul multi channel pipette when subsequently transferring to the plate Add 1 ul of sample or diluted antigen standard to the 96 well plate Note Do not mix by pipetting up and down since bubbles will form instead centrifuge the plate briefly after all samples and diluted antigen standards have been transferred to the plate Seal the plate with an adhesive plastic film Incubate the plate for 2 h at room temperature This incubation can also be done over night at 4 C 16 Proseek Assay Development kit 4 PRE EXTENSION AND EXTENSION a Thaw the Pre Extension Solution and the Extensi
13. during the antigen affinity purification step Even if the supplier states PBS content it is likely that there are residual amounts of primary amines Antibodies from R amp D Systems are suitable to conjugate directly with Proseek Probemaker and do not require dialysis Olink recommends the following standard procedure for buffer exchange Pre equilibrate an illustra MicroSpin G 50 Columns spin column GE Healthcare Art no 27 5330 01 with 1xPBS by first spinning the column at 3000 rpm for 1 min then add 400 ul of 1xPBS and spin again for 1 min and repeat 4 times Place the column in a new microfuge tube Add your antibody 25 50 ul to the column and spin again for 2 min at 3000 rpm The concentration of the collected antibody should be verified by OD 1 mg ml should have an OD 280 nm of 1 4 Concentrating low concentration antibodies prior to Proseek Probemaker conjugation is not recommended unless you have large milligram amounts since losses are very high with filter type concentrations 5 2 SAMPLE MATERIAL Proseek has so far shown to be successful with the following sample types Serum EDTA plasma Citrate plasma Cerebrospinal fluid CSF Note Do not use heparin treated plasma since it will inhibit the real time PCR reaction b 10 Proseek Assay Development kit 5 3 VALIDATION OF ASSAY To be able to properly evaluate your results it is advisable to do a simple validation of your assay e g by ass
14. eek probe A Proseek Probemaker B to create Proseek probe B Proseek Assay Reagents to perform the assay 4 1 1 PROSEEK PROBEMAKER Proseek Probemaker enables conjugation of an antibody to Oligo A or B creating Proseek probe A or B One Proseek Probemaker A or B can be used for only one antibody conjugation Proseek probes from one conjugation is sufficient for approximately 100 000 reactions Each Proseek Probemaker box contains the following Oligo A or B One vial with lyophilized activated oligonucleotide for conjugation of 10 ug antibody at a concentration of 1 mg ml Conjugation Buffer ready to use Reagents for buffering the conjugation reaction Stop Reagent ready to use Reagents for stopping the conjugation reaction Storage Solution A or B ready to use Reagents for preserving the Proseek probe A or B The probe is stable in the Storage Solution for 12 months at 4 4 1 2 PROSEEK ASSAY REAGENTS Proseek Assay Reagents contains all necessary reagents to perform the Proseek Assay protocol Proseek Assay Reagents is available in kit sizes of 100 and 1000 reactions Each Proseek Assay Reagents box contains the following gt Probe Diluent ready to use For dilution and 3 month storage of Proseek probes gt Calibrator Diluent ready to use For dilution of antigen standard and sample if necessary Assay Solution ready to use For combining with Proseek probes to set up incu
15. essing sensitivity dynamic range precision recovery and linearity of dilution 5 3 1 BACKGROUND By adding the Calibrator Diluent only without the antigen you will obtain a value for the background level of your assay 5 3 2 CALIBRATION CURVE SENSITIVITY DYNAMIC RANGE AND PRECISION The calibration curve made from a dilution series of the antigen standard in replicates will give information about the sensitivity dynamic range and precision of your assay Sensitivity or limit of detection is defined as the lowest detectable concentration that significantly exceeds the background level and is calculated as the obtained background concentration 2 standard deviations The precision can be improved by increasing the number of replicates 5 3 3 RECOVERY You will get an indication of the recovery by spiking in the antigen standard at a certain concentration within the dynamic range of the calibration curve into one of your samples Recovery is defined as the ratio of the measured concentration of the spiked in protein in the sample after subtracting the endogenous concentration and the measured amount of the spiked in protein in the Calibrator Diluent expressed as a percentage 5 3 4 LINEARITY OF DILUTION Sample matrix effects can be investigated by spiking in the antigen standard at a high concentration into one of your samples followed by a serial dilution of the spiked in sample The resulting dilution curve should be linear if no
16. ge of the calibration curve Plot the values in a dot plot graph with the log scale concentrations on the and the average Ct values in reverse order on the y axis Use linear regression and display the trend line the equation y kx m and the value in the graph Use the formula 10 to back calculate the concentration of each Ct value in the calibration curve and your samples VEGF 254 264 274 S H 284 29 4 o lt 304 PEN Fig 14 Calibration curve of VEGF 0 1 1000 pM 314 with concentrations converted to log10 scale on 324 y 1 7047 30 515 the xaxis and the average Ct value in reverse 0 9939 order of duplicates on the y axis Standard 33 linear regression is used 2 1 0 1 2 3 4 Concentration VEGF log pM 20 Proseek Assay Development kit 9 Tips and tricks 9 1 PRECISION The use of appropriate equipment and pipetting technique are crucial in order to obtain high precision and low standard deviation between replicates using well calibrated pipettes for small volumes fine and well attached pipette tips and a consistent and accurate pipetting technique are of utmost importance Increasing the number of replicates will also improve the precision 9 2 SENSITIVITY The sensitivity of your assay may be increased by diluting the Proseek probes stored in the Probe Diluent at 650 pM slightly further e g another 1 2 dilution in Probe Diluent before preparing the probe m
17. link AB is not liable for property damage personal injury or economic loss caused by this product The following trademarks are owned by Olink AB Olink Olink Bioscience Proseek Duolink and PLA This product is covered by several patents and patent applications including US 6 511 809 US 7306 904 and related US and foreign patents This product is sold under license from PHRI Properties Inc and may be used under PHRI Properties patent rights outside the field of human in vitro diagnostics DreamTaq is a trademark of Fermentas UAB a wholly owned subsidiary of Thermo Scientific Inc The oligonucleotide labelling components in the Proseek Probemaker kit utilise Lightning Link technology and are provided under license from Innova Biosciences 2011 Olink AB All third party trademarks are the property of their respective owners Olink Bioscience L N K Dag Hammarskj lds v 52B SE 752 37 Uppsala Sweden BIOSCIENCE www olink com 0791 v1 0 2011 05 04
18. on Solution vortex and quick spin Pre heat the thermal cycler to 37 Make sure you have the following Extension program ready in the thermal cycler gt Polymerization 20 min 37 C gt Inactivation of enzyme 10 min 85 C gt Cooling e 4 C b Prepare a Pre Extension master mix by mixing the Pre Extension Solution 10x with high purity water according to the table below and vortex briefly 76 ul of Pre Extension master mix is needed per reaction prepare an excess volume to account for pipetting losses 1 reaction ul j reactions ul Pre Extension Solution 10x 6 High purity water AE m Total volume of Pre Extension master mix 76 Prepare an Extension master mix mixing the Extension Solution 10x and the Extension Polymerase 5 U ul with high purity water according to the table below and vortex briefly Use a reezing block when removing the Extension Polymerase from 20 C 20 ul of Extension master mix is needed per reaction prepare an excess volume to account for pipetting losses Treaction u reactions pl Extension Solution 10x 2 Extension Polymerase 5 U ul High purity water 9 Total volume of Extension master mix 20 d Place the plate from in the pre heated thermal cycler Add 76 yl of the Pre Extension master mix to each well and incubate for 5 min at 37 without sealing the plate e Transfer 20 ul of the Extension maste
19. r mix to each well of the plate Seal the plate with an adhesive plastic film The total volume in each well is now 100 ul f Start the thermal cycler Extension program and use a heated lid Polymerization 100 ul 20 min 37 gt Inactivation of enzyme 100 ul 10 min 85 C gt Cooling e 4 C If not being able to run real time PCR immediately after the Extension the plate can be stored at 4 C for 24 Proseek Assay Development kit 17 5 REAL TIME PCR a b d e f 9 Thaw the Real Time PCR Solution vortex and quick spin Note The real time PCR instrument should be turned on at least 30 min before starting the amplification Prepare a Real Time PCR master mix by mixing the Real Time PCR Polymerase 1 U u with the Real Time PCR Solution according to the table below and vortex briefly Use a freezing block when removing the Real Time PCR Polymerase from 20 6 ul of Real Time PCR master mix is needed per reaction prepare an excess volume to account for pipetting losses 1 reaction ul reactions ul Real Time PCR Solution 5 8 Real Time PCR Polymerase 1 U ul eee eet Total volume of Real Time PCR master mix 6 Transfer 6 ul of the Real Time PCR master mix to each well of a new optical 96 or 384 well plate Add 4 ul of each extension product from step 4f to the plate Seal the plate with an optical adhesive plastic film and centrifuge
20. re use Ready to use solution Probe Diluent store at 20 C Thaw vortex and quick spin before use b Ready to use solution Assay Solution store at 20 C gt Thaw vortex and quick spin before use b Ready to use solution Pre Extension Solution 10x store at 20 C b Thaw vortex and quick spin before use gt Dilute 1 10 in high purity water immediately before use Extension Solution 10x store at 20 C gt Thaw vortex and quick spin before use gt Dilute 1 10 in high purity water immediately before use Extension Polymerase 5 U ul store at 20 C gt Extension Polymerase should be kept at 20 C at all times Use a freezing block 20 C when removing the enzyme from the freezer gt Quick spin the Extension Polymerase before adding to the Extension Solution and high purity water at a 1 100 dilution immediately before use Ensure that the Extension master mix is thoroughly vortexed before addition to the samples Real Time PCR Solution store at 20 C gt Thaw vortex and quick spin before use b Ready to use solution Real Time PCR Polymerase 1 U l store at 20 C gt Real Time PCR Polymerase should be kept at 20 C at all times Use a freezing block 20 C when removing the enzyme from the freezer gt Quick spin the Real Time PCR Polymerase before adding to the Real Time PCR Solution at a 1 30 dilution immediately before use Ensure that the Real Time P
21. s StepOne and Stratagene MX3000P 4 4 SOFTWARE FOR ANALYSIS The result from a Proseek experiment is a result file with Ct values obtained from the real time PCR instrument This result file can easily be exported to a spreadsheet program e g Microsoft Excel where average Ct ACt and standard deviations can be calculated and further converted to concentrations Proseek Assay Development kit 9 5 Assay considerations 5 1 ANTIBODIES Your choice of antibody or antibodies is crucial when setting up the Proseek assay For conjugation with Proseek Probemaker you can use either one single polyclonal batch or two matched monoclonals The antibody or antibodies should meet the following criteria gt If a polyclonal antibody is used it must be raised against the whole native protein or at least large fragments not peptides and be antigen affinity purified not IgG fraction gt antibody must have a concentration of 1 mg ml 10 ul is needed per conjugation gt antibody has to be in an amine free buffer ideally PBS Low levels of BSA 0 1 trehalose 5 and sodium azide 0 0296 are tolerated If you are unsure about the composition of the buffer your antibodies are stored in Olink strongly recommends dialysis or buffer exchange prior to conjugation Antibodies from various sources and suppliers vary in residual primary amine content often in the form of remaining glycine from the acidic elution of the antibody
22. tore the few ul that are left of the reaction in the vial at 4 if you would like to verify the conjugate with gel electrophoresis 10 Incubate for 30 min at room temperature 11 The conjugation is now completed and your Proseek probe is stable in the Storage Solution for 12 months at 4 Write the name of the antibody that was conjugated on the new label Probe A or Probe B 130 nM and put it over the old label Note Stability may vary between antibodies 14 Proseek Assay Development kit 7 2 PROSEEK ASSAY PROTOCOL The following Assay protocol describes how you perform the Proseek assay in a 96 well microplate but you can as well use test tubes PCR type For the real time PCR detection you will need either an optical 96 or 384 well plate or optical test tubes It is advisable to use a multi channel pipette in the reagent transfer steps when it is possible For loading the multi channel pipette you can transfer the master mix to an 8 or 12 tube strip Change pipette tips between sample and reagent transfer steps to avoid cross contamination There is always a loss of volume when pipetting and transferring reagents therefore always prepare master mixes for additional reactions This has also been taken into account in the Proseek Assay Reagents where Assay Reagents in kit size of 100 reactions contains reagents for approximately 120 reactions and Assay Reagents in kit size of 1000 reactions contains reagents for
23. xtension reaction 20 C No or inefficient Ensure that the Real Time PCR Polymerase has been stored real time PCR at 20 C amplification Wrong settings on Ensure that correct settings have been used on the real time the real time PCR PCR instrument instrument Real time PCR Run real time PCR replicates of any real time PCR master mix instrument is not not necessarily Proseek reagents CV should be below 8 operating well on a linear scale if not recalibrate the instrument and contact the manufacturer Low signals Protein levels in By spiking in the antigen standard into one of your samples at in samples samples are lower a concentration within the linear range of the calibration curve than the detection you can confirm that a signal over background is detected and limit the lack of signal in the non spiked samples is not caused by any sample matrix effects Protein in sample Dilute your samples further is highly abundant and not within the dynamic range of the calibration curve 22 Proseek Assay Development kit PROBLEM PROBABLE CAUSE SUGGESTED SOLUTION High Incomplete mixing of Ensure that all reagents are thoroughly mixed before applying standard reagents to the reaction plate deviation of replicates Uncalibrated pipettes Ensure that your pipettes are calibrated and that the pipette tips are well attached to the pipettes Uneven pipetting of Ensure that you use a consistent and accurate pipetting
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