User Protocol WideScreenTM Receptor Tyrosine Kinase Assay Kits
Contents
1. e To avoid perforating the filter plate membrane be sure that the probe height on the xMAP system is adjusted correctly Do not touch the membrane with pipet tips For accurate pipetting touch tips to the sides of the filter plate wells Change tips as necessary to prevent cross contamination e Capture Beads contain fluorescent dyes and are therefore light sensitive To avoid photobleaching keep beads in microcentrifuge tubes covered Cover filter plates containing beads with aluminum foil during incubation steps Streptavidin PE solution is also light sensitive protect from light e To prevent fluorescent dye loss do not use organic solvents with capture beads Beads are incompatible with DMSO concentrations gt 1 e Many of the washing and preparation of aliquots steps are done most easily with an 8 channel or 12 channel pipet manual or automatic However for best results use accurate single channel pipets for manipulation of standards and experimental samples e Ifusing multichannel pipets ensure that tips fit correctly Verify volume accuracy and consistancy e To conduct the protocol efficiently prepare reagents for the next step during incubation periods e When calculating the amount of reagents needed during the various steps prepare 10 excess to allow for pipetting error e Run standard dilution series and experimental samples using the same multiplex configuration For instance if a 7 plex of Bead Kits is used2. C USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB500 Rev B 0108 Page 10 of 23 Bead Based Immunoassay Protocol Considerations Before You Begin e Have on hand the 1X Assay Diluent and 1X Wash Buffer that was prepared during the Lysate Preparation protocol e Important guidelines to follow when using filter plates and the vacuum manifold e Excessive vacuum will cause the filter plate membrane to perforate Adjust the manifold using a non filter ELISA or tissue culture plate ensuring that the vacuum cannot exceed 5 in 127 mm Hg e After adjusting the vacuum place filter plate on the manifold Use fingertips to apply pressure evenly across the plate The liquid should drain in 2 5 sec e To avoid drying out the beads vacuum only long enough to drain all wells Do not allow drained beads to sit for more than 1 min before rehydrating with buffer e It is critical to remove excess buffer from the underside of the filter plate by tapping it on a paper towel several times before adding samples or reagents This prevents samples from wicking out of the wells during incubation steps For the same reason avoid placing filter plate on an absorbent surface during incubations
3. sonicating in an ultrasonic bath for 10 sec and vortexing again for 5 sec 4 Each well receives a total of 50 pl diluted 1X Capture Beads Determine the total volume of 50X Capture Beads needed per well refer to table above and the volume of 1X Assay Diluent needed to bring the total volume per well to 50 yl Multiply these volumes by the number of test wells to determine the total volumes of each component needed Refer to the table on the next page for example calculations 5 Add the calculated volumes of Capture Beads 50X and 1X Assay Diluent to a microcentrifuge tube Vortex 3 sec Protect from light and store at 4 C until use USA and Canada Tel 800 526 7319 novatech novagen com Germany Tel 0800 100 3496 techservice merckbiosciences de United Kingdom and Ireland All Other Countries UK Freephone 0800 622935 www novagen com Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB500 Rev B 0108 Page 14 of 23 Example Calculations 40 test wells including 10 extra Singleplex or RTK 7 plex premixed User assembled multiplex e g 5 plex Test wells 40 40 Volume Capture Beads 50X 1 pl per well 1 pl each bead per well 5 pl total Volume 1X Assay Diluent 49 yl per well 45 pl per well Total Volume 1 pl beads per well x 40 wells 5 pl beads per well x 40 wells Capture Beads 50X 40 pl beads 200
4. 4 C until use 3 Wash wells three times with 1X Wash Buffer as described above After the final vacuum filtration tap filter plate on a paper towel to remove any buffer on the underside Immediately add 100 pl 1X Streptavidin PE solution to each well Incubate for 45 min at room temperature on a platform plate shaker 750 rpm Protect from light 6 Optional Add 30 pl fixation solution to each well 0 2 paraformaldehyde in TBS not provided in the kit Incubate for 5 min at room temperature on a platform plate shaker 750 rpm Protect from light Note Fixation will improve well to well assay reproducibility 7 Wash wells three times with 1X Wash Buffer as described above After the final vacuum filtration tap filter plate on a paper towel to remove any buffer on the underside 8 Immediately add 120 pl 1X Assay Diluent to the beads in each well To fully resuspend beads before running samples on the Luminex system incubate for 3 5 min on a platform plate shaker Protect from light 9 Analyze samples with a Luminex xMAP system according to the manufacturer s instructions USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB500 Rev B 0108 Page 17 of 23 Flowchart
5. ACS StarStation Bio Plex Manager or comparable e Vacuum manifold for filter plates Millipore Cat No MAVMO960R e 96 well plate platform shaker such as IKA MTS4 e BCA protein assay kit EMD Cat No 71285 USA and Canada Tel 800 526 7319 novatech novagen com All Other Countries www novagen com novatech novagen com Germany Tel 0800 100 3496 techservice merckbiosciences de United Kingdom and Ireland UK Freephone 0800 622935 lreland Toll Free 1800 409445 customer service merckbiosciences co uk User Protocol TB500 Rev B 0108 Page 6 of 23 e Polypropylene microcentrifuge tubes e 15ml and 50 ml polypropylene centrifuge tubes e Microcentrifuge e Vortexer e Ultrasonic bath such as Cole Parmer EW 08849 optional e Multichannel pipet optional e Fixation solution 0 2 paraformaldehyde in TBS optional e Syringe tip filter 0 45 pm and syringe or 96 well filter plate e g Millipore MSBVN6510 and 96 well collector plate e Tris buffered saline TBS 10 mM Tris pH 7 5 150 mM NaCl Growth of Cell Lines Considerations Before You Begin e Growth rate and requirements for optimal growth vary considerably between cell lines even the same cell line will grow differently in different laboratories The following conditions are intended as general guidelines only e Cells maintained in culture for long periods of time tend to exhibit slower growth rates and become refractory to stimulation conditions
6. Do not use organic solvents in the immunoassay as they will damage beads irreversibly Beads are falling outside the bead region gates due to photobleaching Do not use expired beads Do not expose the beads to ambient light for gt 10 min Avoid intense light Fluidics system is not running properly Confirm that the waste container is not full the sheath fluid is not empty and the SD fluidics module is turned on Check system calibration using approved calibration beads Verify correct system pressure Confirm that the system is free of air or particulate buildup Follow maintenance steps described in the instrument user manual An immunoassay reagent is used up Solutions were not prepared or used as described in the protocol Confirm correct buffer dilutions and use If additional Wash Buffer is needed TBST 10 mM Tris pH 7 5 150 mM NaCl 0 05 Tween 20 may be substituted If additional Assay Diluent is needed 10 mM Tris pH 7 5 225 mM NaCl 0 05 Tween 20 1 BSA may be substituted If additional 96 well filter plates are required we recommend Millipore Cat No MSBVN1210 High coefficients of variance CVs between replicates Cells grown in 96 well plates show well to well variability To avoid edge effects don t plate cells in outermost wells of plates Plate cells uniformly Add lysis reagents accurately Do not dislodge adherent cells during pre lysis wash steps If necessary dec
7. High throughput compound library screening USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk Extracellular domains Intracellular O domain OF Oe Capture User Protocol TB500 Rev B 0108 Page 4 of 23 Membrane bound RTK Solubilized RTK xMAP Assay Analyze Data AY l Detection s a ntibody Pa Ps eS antibod t Total RTK assay Quantitate RIJ be pA gt MFI a 1 Detergent mediated cell lysis soe Si O 0000P000000 r 00000 gt I n e 7N 1 a wae gt lt gt a ean p J gt pTyr assay Figure 1 WideScreen RTK Assays using Luminex xMAP Technology The WideScreen RTK Assays consist of a series of phosphotyrosine specific RTK assays and companion RTK total protein assays The phosphotyrosine assays utilize RTK specific capture antibodies and a broad spectrum phosphotyrosine detection antibody The total RTK assays which include standards allow the signals from the phosphotyrosine assays to be compared to the total amount of RTK in the sample Components RTK Bead Kits and Standards are used together for multiplex analysis of cell lysates The WideScreen RTK Total Assay Complete Kit and the WideScreen RTK pTyr Ass
8. Incubate for 20 min at 4 C with occasional vortexing 8 Dislodge and solubilize all adherent cells using a rubber policeman or by repeated pipeting Extracts should be clear and non viscous 9 Clear lysates by filtration Pre wet filter or filter plate with TBS then remove all excess buffer For lysates with volume gt 0 2 ml use syringe tip filter pore size 0 45 pm For USA and Canada Tel 800 526 7319 novatech novagen com Germany Tel 0800 100 3496 techservice merckbiosciences de All Other Countries www novagen com novatech novagen com United Kingdom and Ireland UK Freephone 0800 622935 Ireland Toll Free 1800 409445 customer service merckbiosciences co uk User Protocol TB500 Rev B 0108 Page 8 of 23 lysates with volume lt 0 2 ml use a 96 well filter plate e g Millipore MSBVN6510 filtration by centrifugation at 1500 x g for 1 min at 4 C Place a 96 well plate under the filter plate during centrifugation to collect lysates 10 Either proceed immediately to the Bead based Immunoassay Protocol or store aliquots at 70 C Avoid multiple freeze thaw cycles 11 Remove a 50 pl aliquot of each extract for protein quantification by BCA Protein Assay Cat No 71285 Determine the total protein concentration of each extract Note Typical total protein concentrations from cells grown in flasks range from 0 4 mg ml to 2 mg ml depending on the cell line and confluence Typical total protein concentrations from c
9. Prepare 1X Assay Diluent by adding 25 ml 5X Assay Diluent WideScreen Reagent Kit to 100 ml sterile distilled deionized water Store 1X Assay Diluent that will be used within one month at 4 C To avoid microbial growth dispense aliquots of any remaining 1X Assay Diluent and store at 20 C 2 Prepare 1X Wash Buffer by adding 20 ml 10X Wash Buffer WideScreen Reagent Kit to 180 ml sterile distilled deionized water Store at 4 C 3 Calculate the total amount of Extraction Reagent needed Prepare 10 excess to account for pipetting error Format Extraction Reagent T 175 flask 4ml T 75 flask 2 ml T 25 flask l ml 6 well 200 pl well 96 well 120 pl well 4 Prepare the required volume of supplemented Extraction Reagent Per ml Extraction Reagent add 20 ul Phosphatase Inhibitor Cocktail Set V 50X lpl Protease Inhibitor Cocktail IIT 1000X 0 1 pl Benzonase Nuclease Prepare fresh supplemented Extraction Reagent each time cell lysates are made 5 Aspirate and discard culture medium 6 Onice rinse cell monolayer twice with cold Tris buffered saline TBS Remove all TBS For non adherent cells transfer cells to centrifuge tubes centrifuge at 500 x g and wash twice with ice cold TBS 7 Add cold supplemented Extraction Reagent to adherent cells Incubate for 20 min at 4 C with gentle agitation rocking platform or occasional swirling For non adherent cells flick pellet to loosen Add supplemented Extraction Reagent
10. See Step 1 Prepare Titration Buffer on p 10 Resuspend the appropriate lyophilized RTK Total Standards in 120 pl Titration Buffer for each analyte being tested These represent 10x Standard solutions Vortex briefly to ensure all standards are in solution If conducting a singleplex or user assembled multiplex assay add 30 pl of each of the individual RTK Total Standards 10x being assayed to tube 1 Bring the total volume of tube 1 to 300 pl with Titration Buffer and mix well This tube is Dilution 1 of the standard dilution series If using the RTK Total Standards Mix it is only necessary to add 30 pl premixed 7 plex standards to 270 pl Titration Buffer Prepare 4 fold serial dilutions from Dilution 1 as follows Transfer 80 pl from tube 1 to the 240 pl titration buffer in tube 2 mix well Change tips Transfer 80 pl from tube 2 to the 240 pl titration buffer in tube 3 mix well Proceed in similar manner with the serial dilutions through tube 7 The 8th tube contains 240 pl titration buffer only This will serve as the blank control Refer to Appendix B for concentrations of the serially diluted standards USA and Canada Tel 800 526 7319 novatech novagen com Germany United Kingdom and Ireland All Other Countries Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB5
11. de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB500 Rev B 0108 Page 5 of 23 www novagen com WideScreen Performance specifications for the Bead Kits are detailed in the individual Certificates of Analysis available online Each RTK Bead Kit contains sufficient reagents for 100 tests The RTK recombinant standards are used to create standard curves when performing quantitative assays Each individual RTK Total Bead Kit used to quantify a specific RTK target regardless of phosphorylation state includes the appropriate individual recombinant standard Pre mixed RTK recombinant standards are supplied with the RTK Total Assay Complete Kit and the RTK Total 7 Plex The concentration of standards in the standard curves can be found in Appendix B and at www novagen com WideScreen Each recombinant standards mix or individual standard contains reagents sufficient to generate eight singleplex or multiplex standard curves or four standard curves in duplicate Cell Extraction Kit The Cell Extraction Kit contains a cell extraction reagent that releases soluble and membrane proteins efficiently Benzonase Nuclease reduces viscosity due to chromosomal DNA Phosphatase and protease inhibitor cocktails maintain the phosphorylation state and integrity of target proteins during cell extraction The kit contains reagents sufficient to make 20 ml of cell lysate or to process 160 wells of ce
12. for RTK Immunoassay Protocol Prepare Titration Buffer 75 1X Assay Diluent 25 Extraction Reagent e 2000 ul for duplicate standard curves 300 ul for each lysate sample diluted gt 4 fold final Prepare Capture Beads Prepare 7 point Standard Curve Prepare Sample Dilutions Vortex sonicate Capture Beads 50X 30 ul pre mixed RTK Total Standards Dilute lysate four fold in 1X Assay Per well Mix in tube 1 or 30 ul each individual Diluent 1 ul Capture Beads 50X from each RTK Standard in tube 1 Prepare 100 ug ml Dilution in bead kit Bring to final volume of 300 ul with tube 1 10 ug lysate titration 1X Assay Diluent to final volume of titration buffer buffer to final volume of 400 ul 50 ul 240 ul titration buffer in tubes 2 7 150 ul titration buffer in tubes 2 3 4 fold serial dilutions 80 ul from tube 2 fold serial dilutions 150 ul from 1 to tube 2 etc tube 1 to tube 2 etc Pre wet Filter Plate 100 ul 1X Assay Diluent per well vacuum filter after 5 min Capture Bead Analyte Incubation 50 ul diluted 1X Capture Beads to all wells Remove liquid by vacuum filtration Add 100 ul sample dilutions to beads in appropriate wells Add 100 ul standard dilutions to beads in appropriate wells Add 100 ul titration buffer to beads in Blank well s Shake overnight 750 rpm 4 C in darkness Detection Antibody Incubation Per well 1 ul Detection Antibody 100X from each bead kit 1X Assay Diluent to final volume of 100 ul W
13. from tube 2 to the 150 pl titration buffer in tube 3 Mix well Proceed in similar fashion with the serial dilutions through tube 4 5 These dilutions will result in 10 pg 5 pg 2 5 pg or 1 25 pg total cell protein per assay well respectively refer to figure below titration buffer 40 ug total cell protein to final volume of 400 ul with titration buffer J 10 ug 5 ug 2 5 ug 1 25 ug per well in xMAP assay 150 wr 150 1 50 w oe bisa a 150 yl 150 ul 150 ul V V V final total cell protein Step 4 Prepare Capture Beads Individual RTK Total Bead Kits can be multiplexed in all combinations and individual RTK pTyr Bead Kits can be multiplexed in all combinations However RTK Total Beads and RTK pTyr Beads cannot be multiplexed together because antibodies compete for the same analyte Prepare diluted Capture Beads within 1 h of use 1 Calculate the number of test wells needed allowing 10 extra for pipetting error 2 Note the volume of 50X Capture Beads needed per well based on the assay format In all cases this results in 2000 beads per bead region per well Assay Format Vol Capture Beads 50X needed Singleplex one target 1 pl per well User assembled multiplex 1 pl from each individual Bead Kit per well RTK 7 plex premixed 1 pl per well 3 Thoroughly resuspend each vial of Capture Beads 50X by vortexing for 10 sec
14. inhibitor in DMSO according to the manufacturer s instructions Prepare inhibition medium by diluting the inhibitor stock to the desired concentration in 5 ml tissue culture medium lacking FBS For mock inhibitions prepare serum free tissue culture medium lacking inhibitors but including an equivalent volume DMSO 2 Following serum starvation remove medium Replace with inhibition medium or mock inhibition medium Immediately return cells to incubator 3 Incubate at 37 C and 5 CO for 1 h 4 Prepare induction medium by diluting all growth factor stocks to a final concentration of 200 ng ml in tissue culture medium lacking FBS refer to table on p 19 This results in a 2X solution Growth factors can be added separately with each of the following added to a separate T 75 flask 6 flasks total EGF IGF HGF PDGF A B VEGF Vanadate for stimulation of Tie 2 Alternatively all six growth factors can be added simultaneously to one T 75 flask In either case use 5 ml induction medium per T 75 flask For mock inductions prepare tissue culture medium lacking FBS and growth factors 5 Add 2X induction medium or mock induction medium directly to inhibitor treated cells Immediately return cells to incubator 6 Incubate at 37 C and 5 CO for 10 min Note Phosphorylation of many signaling pathway proteins peaks at 5 10 min followed by rapid dephosphorylation 7 Extract cells immediately according to the Ly
15. lysate samples based on the protein quantification values previously determined using BCA assay For example if the original sample concentration was 1 6 mg ml the dilution results in 400 pg ml Note If desired cell extracts can be further diluted to ensure more accurate signal quantification In this case follow the optional steps below Steps 3 5 within this section A range from 1 10 ug total cell protein per assay well is usually optimal 3 Label four microfuge tubes In tube 1 mix the four fold diluted lysate and titration buffer to a final volume of 400 pl and final protein concentration of 100 pg ml 10 pg well later in the assay For example if the four fold diluted extract has a total protein concentration of 400 pg ml mix 100 pl diluted extract with 300 yl titration buffer USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 novatech novagen com Tel 0800 100 3496 techservice merckbiosciences de UK Freephone 0800 622935 Ireland Toll Free 1800 409445 customer service merckbiosciences co uk www novagen com novatech novagen com User Protocol TB500 Rev B 0108 Page 13 of 23 Note 4 If additional dilutions of the extract are desired prepare three additional 2 fold dilutions of the cell extract as follows Add 150 il titration buffer to tubes 2 3 and 4 Transfer 150 pl from tube 1 to the 150 pl titration buffer in tube 2 and mix well Change tips Transfer 150 jl
16. 00 Rev B 0108 Page 12 of 23 Table 2 Serial dilution of pre mixed RTK recombinant standards 7 Plex Tube Volume Standard Volume Final Dilution Titration Concentration Buffer 1 30 ul RTK Total Standards Mix 7 Plex 10X 270 ul 2 80 ul from tube 1 240 ul 3 80 ul from tube 2 240 wl 4 80 ul from tube 3 240 ul See Appendix B 5 80 ul from tube 4 240 wl 6 80 ul from tube 5 240 ul 7 80 ul from tube 6 240 ul 8 BLANK None 240 ul 0 Table 3 Example of serial dilution of five individual RTK Total Standards user assembled multiplex Tube Vol Standard Volume Final Dilution Titration Concentration Buffer 1 5 x 30 ul of each individual RTK Standard 10X 150 pl 150 ul total volume 2 80 ul from tube 1 240 ul 3 80 ul from tube 2 240 ul 4 80 ul from tube 3 240 pl See Appe die B 5 80 ul from tube 4 240 ul 6 80 ul from tube 5 240 ul 7 80 ul from tube 6 240 ul 8 BLANK None 240 ul 0 Step 3 Prepare Sample Dilutions Notes Thaw and if applicable dilute samples within 1 h of use Avoid multiple freeze thaw cycles 96 well samples can be diluted 4 fold with 1X Assay Diluent in the immunoassay plate later in the protocol see Step 5 Combine Capture Beads with Analytes on p 13 1 Dilute lysate samples four fold in 1X Assay Diluent e g 100 pl lysate with 300 pl 1X Assay Diluent Mix well 2 Calculate the protein concentration of the four fold diluted
17. 3800 nl Remove liquid from filter plate by vacuum filtration 6 Add 100 pl 1X Wash Buffer to each well Remove liquid by vacuum filtration Repeat wash and filtration steps twice more for a total of three washes Tap filter plate on a paper towel to remove any buffer on the underside Note Do not allow the beads to dry out Vacuum only long enough to remove all liquid Add the next solution immediately after tapping filter plate on a paper towel 7 Immediately add 100 ul 1X Detection Antibody solution to each well 8 Incubate for 1 h at room temperature on a platform plate shaker 750 rpm Protect from light Note Turn on the Luminex xMAP system The lasers require a 30 min warm up period USA and Canada Tel 800 526 7319 novatech novagen com All Other Countries www novagen com novatech novagen com Germany Tel 0800 100 3496 techservice merckbiosciences de United Kingdom and Ireland UK Freephone 0800 622935 Ireland Toll Free 1800 409445 customer service merckbiosciences co uk User Protocol TB500 Rev B 0108 Page 16 of 23 Step 7 Add Streptavidin Phycoerythrin PE Note Prepare 1X Streptavidin PE solution within 30 min of use 1 Calculate the total volume of 1X Streptavidin PE solution required 100 pl is needed for each test well 2 Prepare the calculated volume of 1X Streptavidin PE solution by diluting Streptavidin PE Concentrate 1 100 in 1X Assay Diluent Vortex 3 sec Protect from light and store at
18. 5 novatech novagen com customer service merckbiosciences co uk User Protocol TB500 Rev B 0108 Page 19 of 23 Alternative 1 Stimulation of Cell Lines in absence of inhibitor treatment Note Have all reagents for cell extraction ready before inducing cells 1 Prepare induction medium by diluting all growth factor stocks to a final concentration of 100 ng ml in tissue culture medium lacking fetal bovine serum FBS refer to table on p 19 This results in a 1X solution Growth factors can be added separately with each of the following added to a separate T 75 flask 6 flasks total EGF IGF HGF PDGF A B VEGF Vanadate for stimulation of Tie 2 Alternatively all six growth factors can be added simultaneously to one T 75 flask In either case use 5 ml induction medium per T 75 flask For mock inductions prepare tissue culture medium lacking FBS and growth factors 2 Following serum starvation remove medium Add 1X induction medium or mock induction medium to starved cells Immediately return cells to incubator 3 Incubate at 37 C and 5 CO for 10 min Note Phosphorylation of many signaling pathway proteins peaks at 5 10 min followed by rapid dephosphorylation 4 Extract cells immediately according to the Lysate Preparation protocol p 7 Alternative 2 Inhibition and Subsequent Stimulation of Cell Lines Note Have all reagents for cell extraction ready before inhibiting and inducing cells 1 Reconstitute
19. In general cell lines passaged lt 15 times are recommended e See Supplementary Protocols on p 18 for sample protocols for stimulation with growth factors in presence or absence of RTK inhibitors Protocol for Growth of Cell Lines 1 Culture cells in T 75 flasks until steady growth is established Most cell lines will tolerate a split of 1 10 1 20 without slowing their growth rate 2 Culture adherent cells until they approach a confluent monolayer or suspension cells until they approach 10 cells per ml Slower growing cell lines such as A431 may initially take up to a week to approach confluency 3 Plate cells using the following table as a general guide Harvest cells for lysate preparation after 2 or 3 days depending on whether the cells are serum starved overnight before harvesting Table 1 Approximate Cell Numbers for Seeding Cell Lines Cell Line T 75 Flask 6 well Plate 96 well Plate per or 10cm Dish per well well A431 2 0 x 10 2 8x 10 4 0 x 10 HeLa 1 2 x 10 1 7x10 1 5 x 10 HepG2 4 8 x 10 6 8 x 10 8 0 x 10 HT29 2 4 x 10 3 4 x 10 3 0 x 10 HUVEC 1 5x 10 2 0 x 10 not recommended NHDF 1 5x 10 1 5 x 10 not recommended SK Br 3 2 0 x 10 2 8 x 10 3 0 x 10 Jurkat 1 0 x 10 1 4 x 10 1 5 x 10 HUVEC and NHDF cells plated with these cell numbers are serum starved after 6 days and lysed after 7 days Note If cells are grown in 96 well pl
20. Total singleplex one target 1 pl per well User assembled RTK pTyr 1 pl per well multiplex User assembled RTK Total 1 pl from each individual Bead Kit per multiplex well RTK pTyr 7plex premixed 1 pl per well RTK Total 7plex premixed 1 pl per well 3 Each well receives a total of 100 pl diluted 1X Detection Antibody solution Determine the total volume of 100X Detection Antibodies needed per well refer to the table above and the volume of 1X Assay Diluent needed to bring the total volume per well to 100 pl Multiply these volumes by the number of test wells to determine the total volumes of each component needed Refer to the table below for example calculations 4 Add the calculated volumes of Detection Antibodies 100X and 1X Assay Diluent to a microcentrifuge tube Vortex 3 sec and store at 4 C until use Example Calculations 40 test wells including 10 extra Singleplex or User assembled RTK Total RTK 7plex multiplex premixed e g 5 plex Test wells 40 40 Volume Detection 1 pl per well 1 pl each Antibody per well 5 pl Antibodies 100X total Volume 1X Assay Diluent 99 jl per well 95 pl per well Total Volume 1 pl Antibody per well x 5 pl Antibodies per well x 40 Detection Antibodies cabs wels 100X 40 pl Detection 200 pl Detection Antibodies Antibody 40p1 ea Total Volume 99 yl per well x 40 wells 95 pl per well x 40 wells 1X Assay Diluent 3960 pl
21. aS Novagen WideScreen Receptor Tyrosine Kinase Assay Kits Table of Contents ADOUE TMG KITS seinien nea aa a Ane taand heats 3 Overview 3 Components 4 Components and Storage 5 Additional Reagents and Equipment Required 5 Growin OF Cell INES sesers a RAR 6 Considerations Before You Begin 6 Protocol for Growth of Cell Lines 6 Lysate Preparation issirinkti arauei iaaea iieii iita 7 Considerations Before You Begin 7 Lysis Protocol for Cell Lines 7 Flowchart for RTK Lysate Preparation cccccscscssesecsseescsecseeseesees 9 Bead Based Immunoassay Protocol c cccccceccsscssseseescsesseeesseeeeeees 10 Considerations Before You Begin 10 Step 1 Prepare Titration Buffer 10 Step 2 Prepare Standard Dilution Series 11 Step 3 Prepare Sample Dilutions 12 Step 4 Prepare Capture Beads 13 Step 5 Combine Capture Beads with Analytes 14 Step 6 Add Detection Antibodies 14 Step 7 Add Streptavidin Phycoerythrin PE 16 Flowchart for RTK Immunoassay Protocol c cccecccceseseeesseeseeeees 17 Collecting Data and Data Analysis c cccccsscccscsscssssesecscsscecsesseseesens 18 Data Acquisition 18 Generation of Standard Curves and Quantitation of Experimental Samples 18 Supplementary Protocols ccccccccesescccsscsscscsscsececssesecscsscsecscsscseeeees 18 Considerations Before You Begin 18 Alternative 1 Stimulation of Cell Lines in absence of inhibitor treatment 19 Alte
22. ant instead of aspirating liquid and tap plate on paper towels If cells become less adherent during overnight serum starvation shorten the serum starvation step to 4 h A gradual drop in signal strength as many samples are read on the xMAP system Group samples such that those being compared directly including replicates are not being read with a long delay in between Use 0 2 paraformaldehyde in TBS to covalently fix PE to bead surfaces Lysates assayed at different times show assay to assay variability Generate standard curves carefully using at least duplicate dilutions series to increase inter assay precision Fully resuspend standards and lysate samples by thawing to room temperature and vortexing carefully Sample measurements not falling on the standard curve Dilution of digested lysate is too low or too high If values are higher than the standard curve dilute samples further in titration buffer Signal strength may be boosted by increasing lysate protein concentration by lysing cells at a higher confluence or by using less Extraction Reagent Standard curve and background values increased due to multiplexing The standard curves of some assays shift slightly upon multiplexing Therefore for accurate quantitation the same multiplex of assays must be prepared when comparing standard curves and experimental samples Target concentration is below detection Ensure that stimulation
23. ash amp vacuum filter plate 3 times 100 pl 1X Wash Buffer per well Add 100 ul diluted 1X Detection Antibody mix to each well Shake 1 h 750 rpm room temperature in darkness Streptavidin PE Incubation Per well 1 yl Streptavidin PE Concentrate 99 ul 1X Assay Diluent Wash amp vacuum filter plate 3 times 100 ul 1X Wash Buffer per well Add 100 ul diluted Streptavidin PE to each well Shake 45 min 750 rpm room temperature in darkness Optional Add 30 ul fixation solution to each well 0 2 paraformaldehyde in TBS not provided in the kit Shake 5 min 750 rpm room temperature in darkness Analysis Wash amp vacuum filter plate 3 times 100 ul 1X Wash Buffer per well Add 120 ul 1X Assay Diluent to each well Shake 5 min 750 rpm room temperature in darkness Run on Luminex system Low Gain RP1 setting BioPlex DD Gate 7500 18500 Luminex Sample size 50 ul Collect 100 events per bead region Timeout 30 sec USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB500 Rev B 0108 Page 18 of 23 Collecting Data and Data Analysis Data Acquisition For detailed instructions on the operation of Luminex systems refer to the user guide for your specific
24. ates plate extra wells for determining total protein concentration of the lysates USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB500 Rev B 0108 Page 7 of 23 If cells will be stimulated prior to extraction serum starve them for 4 16 h before stimulation See Supplementary Protocols on p 18 for sample protocols for growth factor stimulation in presence or absence of inhibitor treatment Note Lysate Preparation Note 4 Prepare lysates when cell density is high but cells are still growing logarithmically For adherent cells this is typically a monolayer that is 80 confluent For suspension cells this is typically a density of 0 5 1 0 x 10 per ml Considerations Before You Begin Lyse induced and uninduced cells at the same time Do not omit steps from the sample preparation protocol All steps are necessary for optimum assay performance If it is important to know the lysate protein concentration from cells grown in 96 well plates prepare additional wells of cells solely for this purpose If using cells grown in 96 well plates avoid plating cells in the outermost rows and columns This minimizes cell growth edge effects Lysis Protocol for Cell Lines 1
25. ay Complete Kit contain sufficient reagents to run 96 test wells For maximum flexibility and user defined multiplex assay configuration components of the WideScreen RTK Total Assay Complete Kit and the WideScreen RTK pTyr Assay Complete Kit are available separately All components are necessary for carrying out the RTK bead based assays The RTK Bead Kits and buffers are not compatible with other bead kits and reagents sold by Novagen or other vendors WideScreen RTK Total Assay Complete Kit The WideScreen RTK Total Assay Complete Kit includes the entire set of reagents to run 96 test wells including the RTK Total 7 plex RTK Total Standards Mix Cell Extraction Kit and WideScreen Reagent Kit WideScreen RTK pTyr Assay Complete Kit The WideScreen RTK pTyr Assay Complete Kit comprises the entire set of reagents to run 96 test wells including the RTK pTyr 7 plex Cell Extraction Kit and WideScreen Reagent Kit No standards are included in the pTyr Assay Complete Kit RTK Bead Kits RTK Bead Kits contain antibody coated Capture Beads and biotinylated Detection Antibodies used for target detection via immunoassay sandwiches Bead Kits may be purchased separately or as pre mixed panels Available Bead Kits are listed in Appendix A and at USA and Canada Tel 800 526 7319 novatech novagen com Germany United Kingdom and Ireland All Other Countries Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com techservice merckbiosciences
26. conditions are optimal Screen additional cell lines Target expression may be suboptimal in some cell lines Confirm that antibodies used in the assay recognize target in the species being tested USA and Canada Tel 800 526 7319 novatech novagen com Germany Tel 0800 100 3496 techservice merckbiosciences de All Other Countries www novagen com novatech novagen com United Kingdom and Ireland UK Freephone 0800 622935 Ireland Toll Free 1800 409445 customer service merckbiosciences co uk User Protocol TB500 Rev B 0108 Page 22 of 23 Appendix A RTK Bead Kits Ordering and Storage Information Each Bead Kit contains the following components e 100 pl Capture Bead 50X use 1 pl per test e 100 pl Detection Antibody 100X use 1 pl per test e 120 pl RTK Total Standard s only available in Total Bead Kits Note Individual RTK Total Bead Kits can be multiplexed in all combinations and individual RTK pTyr Bead Kits can be multiplexed in all combinations However RTK Total Beads and RTK pTyr Beads cannot be multiplexed together because antibodies would compete for their respective analyte RTK Total 7 plex Kits Store at 4 C 71924 3 100 tests EGFR Total IGF 1R Total HGFR Total PDGFR Total HER 2 Total VEGFR2 Total Tie 2 Total RTK pTyr 7 plex Kits Store at 4 C 71925 3 100 tests EGFR pTyr IGF 1R pTyr HGFR pTyr PDGFR B pTyr HER 2 pTyr VEGFR2 pTyr Tie 2 pTyr Ind
27. ells grown in 96 well plates range from 0 1 mg ml to 0 5 mg ml USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB500 Rev B 0108 Page 9 of 23 Flowchart for RTK Lysate Preparation Cells Grown in Flasks or 96 well plates Prepare Stocks amp Buffers 1X Assay Diluent Dilute 5X five fold with water 1X Wash Buffer Dilute 10X ten fold with water Prepare Supplemented Extraction Reagent per 1 ml Extraction Reagent Phosphatase Inhibitor Cocktail 20 ul Protease Inhibitor Cocktail Benzonase Nuclease Cell Lysis Discard culture medium Wash cells twice with ice cold TBS Add supplemented Extraction Reagent 4 ml per T 175 flask 2 ml per T 75 flask 1 ml per T 25 flask 200 ul per well of 6 well plate 120 ul per well of 96 well plate Incubate for 20 min on ice Filtration of cell lysates Pre wet filter or filter plate with TBS For lysates with volume gt 0 2 ml use a syringe tip filter pore size 0 45 um For lysates with volume lt 0 2 ml use a 96 well filter plate e g Millipore MSBVN6510 centrifuge 1500 x g 1 min 4 C Determine Total Cellular Protein Concentration e Use BCA Protein Assay Proceed to bead based immunoassay or store lysate aliquots at 70
28. es prepared as per Step 3 Prepare Sample Dilutions on p 12 If additional sample dilutions were prepared optional add 100 pl of these dilutions to bead containing wells If working with samples generated from cells grown in 96 well plates dilute them directly four fold with 1x Assay Diluent in the immunoassay plate Add 75 ul 1X Assay Diluent and 25 ul cell lysate directly to the appropriate wells of the 96 well filter plate For convenience we recommend using a multichannel pipet 6 Incubate overnight at 4 C on a platform plate shaker 750 rpm Use aluminum foil to protect filter plate from light Shorter incubations are possible but will decrease overall signal strength Step 6 Add Detection Antibodies Prepare 1X Detection Antibody solution within 1 h of use 1 Calculate the number of test wells needed allowing 10 extra for pipetting error 2 Note the volume of 100X Detection Antibody needed per well based on the assay format see table on next page USA and Canada Tel 800 526 7319 novatech novagen com All Other Countries www novagen com novatech novagen com Germany Tel 0800 100 3496 techservice merckbiosciences de United Kingdom and Ireland UK Freephone 0800 622935 Ireland Toll Free 1800 409445 customer service merckbiosciences co uk User Protocol TB500 Rev B 0108 Page 15 of 23 Assay Format Volume Detection Antibodies 100X needed RTK pTyr singleplex one target 1 pl per well RTK
29. gen com customer service merckbiosciences co uk User Protocol TB500 Rev B 0108 Page 11 of 23 Note Notes Note Prepare fresh titration buffer for each assay l Calculate the total amount of Titration Buffer needed A minimum of 2000 pl titration buffer is needed to prepare a duplicate standard curve see Step 2 Prepare Standard Dilution Series below A minimum of 300 pl titration buffer is needed for each lysate sample that is diluted more than 4 fold final see optional steps in Step 3 Prepare Sample Dilutions on p 12 Sample Calculation 2 Standard dilution series 2000 ul 30 Diluted lysate samples 9000 ul 30 X 300 ul Make at least 11000 ul titration buffer 2 Prepare the required volume titration buffer by mixing Extraction Reagent from the Cell Extraction Kit and 1X Assay Diluent prepared from the WideScreen Reagent Kit Use a ratio of 25 Extraction Reagent to 75 1X Assay Diluent In the example above take 2750 pl Extraction Reagent 8250 pl 1X Assay Diluent 11000 yl Titration Buffer allowing for additional buffer to account for pipetting error Step 2 Prepare Standard Dilution Series Standards are only available for RTK Total Bead Kits No standards are available for RTK pTyr Bead Kits Prepare fresh diluted standards for each assay and use within 1 h l 5 To prepare duplicate 7 point standard curves label eight microcentrifuge tubes and add 240 pl Titration Buffer to tubes 2 8
30. he WideScreen RTK Assay workflow is shown in Figure 1 on p 4 If expressed in the cell line of interest individual RTK proteins may be present in either inactive or active states depending on availability of the RTK ligand Cellular protein samples are prepared by gentle membrane extraction Extracts are combined with dye labeled Luminex xMAP beads covalently conjugated to capture antibodies specific to each RTK After incubation and several washes the beads are incubated with biotin labeled detection antibodies The resulting bead immobilized immunosandwich is detected with streptavidin phycoerythrin and quantified using a Luminex xMAP instrument Importantly detection antibodies differ between the WideScreen Total RTK assay and the WideScreen pTyr RTK assay In the Total assay the detection antibody recognizes a second epitope on the RTK allowing quantification of RTK levels without regard to phosphorylation state In the pTyr assay the detection antibody is specific to conserved pTyr on all RTKs Relative quantification of RTK phosphorylation expressed in median fluorescence intensity units is possible using the pTyr kit Because antibodies would compete for the same analyte the RTK Total Assays cannot be multiplexed with the RTK pTyr assays Applications of the WideScreen RTK Assay Kits include e Biomarker quantification e Expression profiling e Confirmation of knock down experiments e RTK agonist or antagonist profiling e Pathway analysis e
31. iew Bead based flow cytometric assays enable sensitive precise quantification of analytes within a sample When directed towards protein analytes such assays are essentially ELISAs on a bead Samples are combined with labeled microparticles covalently conjugated to a capture antibody Analytes captured on the beads are identified with detection antibodies and a fluorescent label A major advantage over traditional protein analyte quantification methods such as ELISA is the capacity for multiplexing as bead based assays allow simultaneous quantification of multiple analytes in a small sample volume Receptor tyrosine kinases RTKs are critical regulators of numerous cell signaling pathways and have been implicated in various disease states Ligand binding to the extracellular domain of transmembrane RTKs triggers receptor dimerization and autophosphorylation of an intracellular kinase domain This event ultimately triggers activation of downstream pathway proteins via phosphotyrosine SH2 domain interactions The WideScreen RTK Assay Kits allow quantification of a set of key RTKs including e Epidermal Growth Factor Receptor EGFR e Insulin like Growth Factor 1 Receptor IGF 1R e Hepatocyte Growth Factor Receptor HGFR e Platelet Derived Growth Factor Receptor beta PDGFRB e Human Epidermal Growth Factor Receptor 2 HER 2 e Vascular Endothelial Growth Factor Receptor 2 VEGFR2 e Tyrosine Kinase with Immunoglobulin and EGF Repeats 2 Tie 2 T
32. ividual RTK Total Bead Kits 100 tests EGFR Total Bead Kit Store at 4 C 71928 3 100 tests IGF 1R Total Bead Kit Store at 4 C 71929 3 100 tests HGFR Total Bead Kit Store at 4 C 71930 3 100 tests PDGFR Total Bead Kit Store at 4 C 71931 3 100 tests HER 2 Total Bead Kit Store at 4 C 71932 3 100 tests VEGFR2 Total Bead Kit Store at 4 C 71933 3 100 tests Tie 2 Total Bead Kit Store at 4 C 71934 3 Individual RTK pTyr Bead Kits 100 tests EGFR pTyr Bead Kit Store at 4 C 71935 3 100 tests IGF 1R pTyr Bead Kit Store at 4 C 71936 3 100 tests HGFR pTyr Bead Kit Store at 4 C 71937 3 100 tests PDGFRB pTyr Bead Kit Store at 4 C 71938 3 100 tests HER 2 pTyr Bead Kit Store at 4 C 71939 3 100 tests VEGFR2 pTyr Bead Kit Store at 4 C 71940 3 100 tests Tie 2 pTyr Bead Kit Store at 4 C 71941 3 WideScreen RTK Total Assay Complete Kit 71942 3 1 RTK Total 7 Plex which includes Store at 4 C e RTK Capture Beads Premix 7 Plex e RTK Total Detection Antibody Premix 7 Plex e RTK Total Standards Mix 7 Plex 1 Cell Extraction Kit see p 5 for components Store at 20 C 1 WideScreen Reagent Kit see p 5 for see p 5 for components storage conditions USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Tol
33. l Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB500 Rev B 0108 Page 23 of 23 WideScreen RTK pTyr Assay Complete Kit 1 RTK pTyr 7 Plex which includes e RTK Capture Beads Premix 7 Plex e pTyr Detection Antibody 71943 3 Store at 4 C 1 Cell Extraction Kit see p 5 for components Store at 20 C 1 WideScreen Reagent Kit see p 5 for see p 5 for components storage conditions Note The RTK Bead Kits and reagents are not compatible with other bead kits and reagents sold by Novagen or other vendors Appendix B Dilution Series for Generating Standard Curves The standard curve is used to quantify target proteins found in cell extracts and other analytes Standards are recombinant proteins representing the extracellular domain of target proteins Notes Standards are supplied with RTK Total Bead Kits only No standards are available for RTK pTyr Bead Kits Standard concentrations are assay dependent This is because the linear range and lower limit of each assay depends on assay sensitivity Values shown are the final concentrations in pg ml Standards supplied with Bead Kits for individual Total targets contain only the standard of interest but can be mixed for multiplex analysis RTK Total Standards Mix Final concentrations in the 4 fold serial dilution of the standards Total Total T
34. lls grown in 96 well plates Additional extraction reagent is included for preparation of titration buffer WideScreen Reagent Kit The WideScreen Reagent Kit contains reagents needed for the bead based immunoassays including all buffers a 96 well filter plate a plate sealer and a streptavidin phycoerythrin solution used in the final detection step The kit contains sufficient reagents to perform 96 singleplex or multiplex bead based tests Components and Storage Cell Extraction Kit 71926 3 25 ml Extraction Reagent Store at 20 C 500 pl Phosphatase Inhibitor Store at 20 C Cocktail Set V 50x 25pl Protease Inhibitor Cocktail Store at 20 C Set III 1000x 10 wl Benzonase Nuclease HC Store at 20 C Purity gt 99 250 U l WideScreen Reagent Kit 71783 3 100 ul Streptavidin PE Concentrate Store at 4 C 20 ml 10X Wash Buffer Store at 4 C 25 ml 5X Assay Diluent Store at 4 C lea Polyethylene Plate Sealer Store at room temperature lea 96 well Filter Plate Store at room temperature Components and storage conditions for WideScreen RTK Bead Kits and RTK Assay Complete Kits are described in Appendix A Additional Reagents and Equipment Required e Experimental samples such as cultured cell lines treated with or without stimulant e Luminex xMAP System or comparable such as Bio Plex Suspension Array System e xMAP data analysis software e g Luminex IS
35. membrane Do not use contaminated reagents Timeout limit is set too low Use the recommended settings for acquisition setup first 50 ul sample 100 events per bead 30 sec time out etc However timeout limit can be set higher e g 75 s Data acquisition is slow No beads in the wells or fluidics system is clogged See Low bead counts during data acquisition solutions above Some bead regions being acquired are not in the wells Make sure that the intended beads were added and that the correct bead regions and wells were selected during acquisition setup Attempting to acquire inappropriate bead regions will cause each sample to time out USA and Canada Tel 800 526 7319 novatech novagen com Germany Tel 0800 100 3496 techservice merckbiosciences de United Kingdom and Ireland UK Freephone 0800 622935 Ireland Toll Free 1800 409445 All Other Countries www novagen com novatech novagen com customer service merckbiosciences co uk User Protocol TB500 Rev B 0108 Page 21 of 23 Problem Probable Cause Solution Beads are not falling into the gates properly Beads were not resuspended in 1X Assay Diluent before analysis The setting of the Doublet Discriminator DD gate is buffer specific This gate can be adjusted but 1X Assay Diluent is the buffer recommended for running samples Other buffers may also cause bead aggregation Beads were exposed to organic solvents
36. nd lower limits of detection However it is not necessary to subtract the MFI value of the blank from other measurements and the blank is generally not plotted as part of the curve e Five Parameter Logistic 5PL curve fitting is recommended for modeling data Most ranges of standard curve concentrations are too wide for accurate linear regression analysis Four parameter 4PL equations will often give a good fit but are not ideal because they assume the standard curve is symmetrical e Ifthe signal from an experimental sample exceeds the highest point of the standard curve the concentration of the unknown should not be extrapolated because the non linear shape of the standard curve cannot be accurately modeled past the last measured point In this case samples should be diluted and tested again Supplementary Protocols Considerations Before You Begin Include appropriate positive and negative controls whenever possible Increases in target protein phosphorylation can be demonstrated by comparison to unstimulated cells cells treated with RTK inhibitors or by treating cell extracts with lambda protein phosphatase In this last case Phosphatase Inhibitor Cocktail should not be added to the Extraction Reagent USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 40944
37. otal Total Total Total Total EGFR IGF IR HGFR PDGFRB HER 2 VEGFR2 Tie 2 Molecular Weight kDa 68 48 129 84 96 160 100 Bead Region 21 25 30 43 72 76 80 pg ml pg ml pg ml pg ml pg ml pg ml pg ml Dilution 1 20000 100000 50000 20000 20000 100000 10000 Dilution 2 5000 25000 12500 5000 5000 25000 2500 Dilution 3 1250 6250 3125 1250 1250 6250 625 Dilution 4 313 1563 781 313 313 1563 156 Dilution 5 78 391 195 78 78 391 39 Dilution 6 20 98 49 20 20 98 10 Dilution 7 5 24 12 5 5 24 2 Blank 0 0 0 0 0 0 0 USA and Canada Germany United Kingdom and Ireland Tel 800 526 7319 novatech novagen com Tel 0800 100 3496 techservice merckbiosciences de UK Freephone 0800 622935 Ireland Toll Free 1800 409445 customer service merckbiosciences co uk All Other Countries www novagen com novatech novagen com
38. pl beads 40p11 ea Total Volume 49 yl per well x 40 wells 45 pl per well x 40 wells 1X Assay Diluent 1960 pl 1800 nl Note Note Note Note Step 5 Combine Capture Beads with Analytes 1 Pre wet 96 well filter plate wells with 50 pl 1X Assay Diluent for 5 min Leave dry any wells that will not be used These wells can be used in future assays use the plate sealer for storage With the vacuum manifold apply gentle vacuum 3 in Hg 76 mm Hg to filter plate just until liquid aspiration is complete Tap filter plate on a paper towel to remove any buffer on the underside It is critical to remove excess buffer from the underside of the filter plate before adding samples or reagents Otherwise samples may wick out of the wells during incubation steps For the same reason avoid placing filter plate on an absorbent surface during incubations See Considerations Before You Begin on p 10 for guidelines on using the filter plate vacuum and manifold 2 Vortex 10 sec the diluted Capture Beads solution prepared as per Step 4 Prepare Capture Beads on p 13 Add 50 pl to each well being used Remove liquid from filter plate by vacuum filtration 4 To bead containing wells reserved for the standards add 100 pl from the standard dilutions Dilutions 1 7 blank prepared as per Step 2 Preparing Standard Dilution Series on p 11 5 To bead containing wells reserved for analyzing experimental samples add 100 pl diluted sampl
39. rnative 2 Inhibition and Subsequent Stimulation of Cell Lines 19 Troubleshooting cccccccsssesesesecscessescessensecsessecseeseensessesseceteneeseenees 20 Appendix A RTK Bead Kits Ordering and Storage Information 22 Appendix B Dilution Series for Generating Standard Curves 23 USA and Canada Europe All Other Countries Tel 800 526 7319 France Germany lreland United Kingdom All other Contact Your Local Distributor novatech novagen com Freephone Freecall Toll Free Freephone European Countries www novagen com 0800 126 461 08001003496 1800 409 445 0800 622 935 44 115 943 0840 novatech novagen com techservice merckbio eu www novagen com FOR RESEARCH USE ONLY NOT FOR HUMAN OR DIAGNOSTIC USE User Protocol TB500 Rev B 0108 Page 2 of 23 2008 EMD Chemicals Inc an Affiliate of Merck KGaA Darmstadt Germany All rights reserved The Novagen name and Novagen logo are registered trademarks of EMD Chemicals Inc in the United States and in certain other jurisdictions WideScreen is a trademark of EMD Chemicals Inc Benzonase is a registered trademark of Merck KGaA Darmstadt Germany Bio Plex is a registered trademark of Bio Rad Laboratories Inc Luminex and xMAP are registered trademarks and Luminex 100 IS and Luminex 200 are trademarks of Luminex Corporation By opening the packaging containing this product which contains fluorescently labeled microsphere beads au
40. sate Preparation protocol p 7 USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB500 Rev B 0108 Page 20 of 23 Table 4 Growth factor reconstitution and dilution Final Growth raer Reconstitution Stock solution Concentration in per Cat No Reconstitution Amount ug volume ul ug ml 1X Induction Medium ng ml EGF 324831 10 mM acetic acid 0 1 BSA 200 2000 100 100 HGF 375228 PBS 0 1 BSA 5 50 100 100 IGF 407240 PBS 0 1 BSA 50 500 100 100 PDGF AB 521220 4 mM HCl 0 1 BSA 10 100 100 100 VEGF 676472 PBS 0 1 BSA 10 100 100 100 Vanadate 567540 water 200 mM 10 mM Troubleshooting Problem Probable Cause Solution Lysate is viscous Genomic DNA is not digested Make sure Benzonase Nuclease was added to Extraction Reagent Incubate lysate longer For cell lines with recurring viscosity problems additional Benzonase Nuclease can be added available separately Leaking wells in filter plate Wicking due to adherent drops Tap filter plate several times on paper towel before adding samples or reagents Do not place filter plate on an absorbent surface during incubations If wells leaked during data acquisition i
41. software and instrument General recommendations are given below 1 Using your Luminex system software prepare a Protocol for the assay you will run Enter in information for each Bead Kit target and for the standards samples and controls that will be run The ranges of final concentrations found in the RTK Total Standards Mix 7 plex are shown in Appendix B 2 Select the bead regions used in the assay The bead regions used for the RTK Bead Kits are shown in Appendix B 3 Format the assay plate indicating which wells contain which type of analyte Acquire data using the system settings shown below Software Sample Events per Timeout Doublet CAL2 Gain Size Bead Discriminator Setting Region Luminex 100 IS 50 ul 100 30 sec 7500 18500 default ACS StarStation 50 ul 100 30 sec default default Bio Plex Manager 50 ul 100 default default RP1 Low Generation of Standard Curves and Quantitation of Experimental Samples e Standards are available for all of the RTK Total Bead Kit assays see Appendix B allowing accurate quantification Representative standard curves and assay performance information can be found in the Certificates of Analysis for the individual bead kits e The 7 point standard curves are plotted using Median Fluorescent Intensity MFT as the signal readout Y axis against concentration of standard dilutions X axis Measurements of the blank are useful for assessing background a
42. t may be possible to reacquire these wells Blot underside of the wells and add 120 ul well 1X Assay Diluent Perforation of filter plate membranes Adjust the vacuum setting to lt 5 inches 127 mm Hg Do not touch membranes with pipet tips Filter plate wells not draining under vacuum Vacuum is too low Adjust vacuum setting to 3 5 inches 76 127 mm Hg Clean rubber seals Apply fingertip pressure to filter plate to ensure formation of a good seal Use a plate sealer to cover wells not in use Cell debris clogs membranes Clarify lysates by centrifugation Avoid disturbing pellets when removing supernatant If lysate protein concentration is high dilute further before assaying Low bead counts during data acquisition No beads or wrong beads in the wells See solutions above for leaking wells Verify that the appropriate beads were added at the correct concentration and that the correct bead regions and wells were selected during acquisition setup Luminex fluidics system is clogged Clear system of clogs or air using maintenance steps described in the instrument user manual sanitize alcohol flush probe sonication etc Make sure that the probe height is set correctly Make sure that beads are in suspension by incubating plate for 3 5 min on the platform plate shaker 750 rpm immediately before analysis Microbial growth in buffers can cause beads to stick to the filter plate
43. thorized by Luminex Corporation or using this product in any manner you are consenting and agreeing to be bound by the following terms and conditions You are also agreeing that the following terms and conditions constitute a legally valid and binding contract that is enforceable against you If you do not agree to all of the terms and conditions set forth below you must promptly return this product for a full refund prior to using it in any manner You the buyer acquire the right under Luminex Corporation s patent rights if any to use the product or any portion of this product including without limitation the microsphere beads contained herein only with Luminex Corporation s laser based fluorescent analytical test instrumentation marketed under the name Luminex Instrument The terms and conditions governing EMD Chemicals sale of this product are as indicated on our website www emdbiosciences com USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB500 Rev B 0108 Page 3 of 23 About the Kits Cell Extraction Kit 1 kit 71926 3 WideScreen Reagent Kit 1 kit 71783 3 WideScreen RTK Complete Assay Kits RTK Total Bead Kits See Appendix A RTK pTyr Bead Kits See Appendix A Overv
44. to measure experimental samples the same 7 plex should be used to create the standard dilution series Multiplexing causes slight shifts in some standard curves which will make quantification inaccurate unless experimental samples are measured using the same multiplex e For best overall assay performance lysates are diluted at least 4 fold when incubating with the Capture Beads If desired lysates can be tested at a 2 fold final dilution although this concentration of Lysis Buffer decreases the sensitivity of some Bead Kits If a 2 fold final dilution is used change the titration buffer composition to 50 Lysis Buffer 50 1X Assay Diluent to ensure accurate quantification Final dilutions less than 2 fold are not recommended Step 1 Prepare Titration Buffer Quantitative immunoassays are sensitive to buffer composition Therefore include the same proportion of Extraction Reagent in all dilutions of standards and samples The best overall assay performance occurs when lysates are diluted at least 4 fold when incubated with the Capture Beads Titration buffer as described here 25 Extraction Reagent 75 1X Assay Diluent maintains a 4 fold final dilution of Extraction Reagent in all assay wells USA and Canada Tel 800 526 7319 novatech novagen com Germany United Kingdom and Ireland All Other Countries Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech nova
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