user manual 4
Contents
1. 16 Enzyme titration The second optimization step consists of enzyme titra tion using the optimal substrate and ATP concentra tions determined previously As observed in Figure 5 increasing the concentration of enzyme up to approximately 0 3 nM led to a propor tional signal increase A S B ratio of approximately 31 was obtained when using 0 3 nM of enzyme Above this enzyme concentration a signal decrease was ob served which may reflect either 1 saturation of both beads Acceptor and Donor by an excess of phos phorylated product or 2 competition of biotinylated product binding to the PhosphoSensor Acceptor beads by the kinase itself As a matter of fact some kinases are phosphorylated for activation 700004 gt without staurosporine T 60000 with staurosporine 50000 c2 9 E 40000 ps 8 8 300004 S 200004 T 10000 4 og aa a 1411 10 9 8 7 6 log PKA M Figure 5 Enzyme titration using optimal concentrations of ATP and substrate PKA was titrated using 3 uM of ATP and 0 3 uM of biotinylated substrate a detection time of 1 hour was used Transfer assay format In the transfer assay format a bulk kinase reaction is performed subsequently the reaction mix is diluted before adding the AlphaScreen beads In this format the following concentrations of ATP enzyme and bioti nylated substrate are suggested as a starting point 100 uM 30 nM and 10 uM respectively Af2. integrated later during assay development see section IV E The following example shows preliminary assay develop ment using the commercially available protein kinase A PKA and the biotinylated substrate kemptide All assays were performed at room temperature RT in white Opaque 384 well microplates in a final volume of 26 uL using 2 uL of enzyme 2 uL of biotinylated peptide 2 uL of ATP 10 uL of PhosphoSensor Acceptor beads and 10 uL of Streptavidin Donor beads both acceptor and donor beads were used at a final concentration of 20 ug mL 1 ATP substrate titration The first optimization step consists of titrating both the substrate and ATP concentrations We recommend the matrix depicted in Table III which uses both fixed en zyme 3 nM and beads concentrations 20 yug mL The assay window should be determined by performing the assay in the absence or presence of staurosporine Table III Scheme of preliminary kinase assay development For preliminary assay conditions perform the titration of ATP and substrate using 3 nM of enzyme The assay should be performed in the absence and presence of 10 uM of a generic inhibitor such as staurosporine with at least one concentration of substrate to evaluate assay back ground With 10 uM stauro Substrate Pat cas uM Ps Tan fo eoa mee 0 10 0 3 0 3 EA 1 Pea 3 ee 3 P e Microplates were read after a detection time of either 1 hour Figure 4A or 17 h
3. kinase reac tion buffer Add 2 uL of biotinylated substrate ATP dilution mix di luted in the kinase reaction buffer Incubate 2 hours kinase dependent at room tempera ture Add 2 uL of EDTA diluted in the optimal detection buffer Add 10 uL of PhosphoSensor Acceptor beads diluted 1 90 in the optimal detection buffer Add 10 uL of Streptavidin Donor beads diluted 1 90 in the optimal detection buffer Incubate in the dark for 1 hr at room temperature Read on an AlphaScreen reader Incubate overnight in the dark and read again Note 9 Detection reaction should read after an over night incubation for optimal results 27 4 Protocol for the transfer assay a Enzyme substrate and ATP are mixed together in a total volume of 100 uL of kinase reaction buffer for reagents concentrations see section IV D The mixture is incubated for 2 hours kinase depend ent at room temperature see section IV E EDTA is added to the mixture The kinase reaction mixture is diluted as described in Table V Table V Dilution of the kinase mixture Dilution Volume of dilution Kinase reaction uL buffer ea feaction mix mix O1 o 3 r e arenes a j e M e Dilute PhosphoSensor Acceptor beads 1 100 in the optimal detection buffer in order to get a final concen tration of 20 ug mL Dilute Streptavidin Donor beads 1 100 in the optimal detection bufferin order to get a final concentration of 20 ug mL g Add
4. of the assay to ATP 2 Perform the assay in a transfer assay 3 Perform kinase assay in smaller volume and or detection assay in large volume to increase the dilution of ATP before the detection Kinase assay Perform the assay in a transfer conditions format to evaluate if it is due to interference of ATP and or enzyme Instrument plates Incompatible microplate choice use solid opaque white plates such as PerkinElmer Optiplates Ensure that your reader contains an AlphaScreen reading mode Kinase assay No or improper biotinylation of reagents substrate peptide or protein check extent of biotinylation using the AlphaScreen TruHit kit Cat No 6760627 31 Possible Cause Effect Remedy No signal Kinase assay Verify that the distance between the reagents phosphorylated amino acid and the biotin is at least 20 carbons Protein contains lysine residues near the phosphorylation site target cysteines for biotinylation using maleimide driven coupling reaction Cofactor required for optimal enzymatic activity add the cofactor in the kinase reaction buffer Peptide substrate not sufficient for efficient phosphorylation by the enzyme use the full length or a longer domain as substrate Reagents degradation perform the assay with fresh enzyme and or substrate Detection Non phosphorylated peptide is binding background conditions non specifically to the PhosphoSensor Acceptor beads test differen
5. 000 cg 5 7500004 o 0 CRS 2 500000 4 2 lt 2500004 0 0 10 20 30 40 50 60 time h Figure 7 Kinetics of the detection reaction using the PhosphoSensor Acceptor beads to perform antibody free kinase activity detection The signal to background ratio is defined with or without 10 uM stauro sporine Note 5 The time course of the detection will vary for other peptide or protein substrates 20 G Titration of PhosphoSensor Acceptor beads In general it is recommended to use a final concentration 20 ug mL of AlphaScreen beads per reaction However for kinases which have affinities for ATP in the high micromolar range tolerance of the PhosphoSensor Acceptor beads to ATP can be improved by increasing their concentration in the detection reaction The following table shows an exam ple using phosphorylation of the biotinylated crosstide pep tide substrate by MSK 1 Table III When the concentration of beads was increased to 50 ug mL more than two fold increase in S B was observed Table III Effect of Acceptor beads concentration on tolerance to ATP In this reac tion 0 3 uM of biotinylated crosstide was phosphoryated by 3 nM of MSK 1 in the presence of different concentration of ATP Enzymatic reactions were allowed to pro ceed for 2 hours in the absence max or presence min of staurosporine Detection was performed using different bead concentrations as indicated PhosphoSensor Acceptor beads 20 pg mL 50 ug mL
6. 22 E If no specific signal is observed in the kinase assay perform the assay in a transfer format to eliminate possible interfer ences with the detection reaction A Choose substrate biotinylation strategy Perform substrate biotinylation B Titrate of substrate and phoshorylated substrate if available C1 Specific window with C2 Specific window high observed non specific background with low non specific background Determine optimal buffer D Titrate kinase to reduce non specific reagents using all in one binding well format Signal generated No signal generated Determine MgClz requirements g Include EDTA to terminate kinase activity g Determine optimal detection time E Perform transfer assay to eliminate possible interferences Figure 9 Scheme of assay development using the AlphaScreen PhosphoSensor Kit 23 Vi DETAILED PROTOCOLS A Titration of phosphorylated versus non phosphorylated substrate Protocol suggested for optimizing buffer conditions using phosphorylated and non phosphorylated substrates see section III C the following protocol will allow the titration of both the phosphorylated and non phosphorylated peptides For performing more than two titration curves increase the volume of each reagent 1 Prepare reaction buffer 2 Prepare detection buffer 10 mM Tris pH 7 0 100 mM NaCl 0 1 Tween 20 3 Prepa
7. ATP uM min max S B min max S B 0 21395 43965 2 1 262727 227959 1 30 6348 54727 9 19214 447904 23 100 6315 23108 4 15102 113449 8 21 SUMMARY OF ASSAY DEVELOPMENT Figure 9 illustrates the different steps that should be under taken in the development of a kinase assay using the AlphaScreen PhosphoSensor Kit In summary the development of a kinase assay involves the following steps A Identify substrate and perform the appropriate biotinylation see section IV A B Perform titration of the biotinylated substrate together with the phosphorylated version of the substrate if available to evaluate the expected signal window see section IV C C1 If a high background signal is observed due to non specific binding of the biotinylated substrate optimize the detection buffer Note 6 Be aware that the presence of ATP in the detection reaction may alleviate the non specific binding of the substrate see section IV D Thus we do not use plus or minus ATP as an indication of the as say window C2 If the peptide titration assay generates a specific signal window continue with the kinase assay development using an all in one well assay format see sections IV D E and F D Titrate substrate ATP and enzyme using an all in one well kinase assay format If a specific signal is observed in the kinase assay proceed with assay optimization see sections IV D E and F
8. PERKINELMER LIFE AND ANALYTICAL SCIENCES j gt PerkinElmer precisely USING THE ALPHASCREEN PHOSPHOSENSOR KIT CATALOG NUMBERS 6760307D 6760307M 6760307R For Laboratory Use Only Research Chemicals for Research Purposes Only Precautions e AlphaScreen beads are light sensitive All assays using the AlphaScreen beads should be performed under sub dued laboratory lighting of less than 100 lux Alternatively green filters Roscolux filters 389 from Rosco or equiva lent can be applied to light fixtures Any incubation of AlphaScreen beads should be performed in the dark Plates can be covered by an opaque microplate to mini mize the effect of light e Due to the small volumes used in the assay it is recom mended that the plates be covered with TopSeal A adhe sive sealing film to reduce evaporation during incubation periods PerkinElmer Inc Cat No 6005185 The assay can be read with the TopSeal A film in place e The PhosphoSensor Acceptor beads contained in this kit may slightly aggregate with time This is normal It is ad vised to vortex the beads prior to use e Beads should be stored in the dark at 4 C lt onmo OWS Vi VII TABLE OF CONTENTS BEFORE STARTING INTENDED USE PRINCIPLE OF THE ASSAY ASSAY DEVELOPMENT Deciding on substrate configuration Deciding on assay format Titration of biotinylated phosphopeptide and or non phosphorylated peptide substrate Evaluat
9. aScreen PhosphoSensor Kit is intended to perform antibody free detection of phosphorylated protein or peptide PRINCIPLE OF THE ASSAY In cells protein kinases mediate the phosphorylation of a vari ety of different protein substrates in the presence of ATP Kinases catalyze the reversible addition of phosphate mole cules to tyrosine serine and threonine residues There are sev eral commercially available antibodies that recognize phospho tyrosine residues with high affinity However such generic anti bodies are not currently available for phosphoserine and phos phothreonine The AlphaScreen PhosphoSensor Kit allows de tection of the phosphorylation of tyrosine serine and threonine residues without the need for such sequence specific antibody The principle of the assay is illustrated in Figure 1 In this as say the kinase driven addition of a phosphate group to a bioti nylated substrate will result in the simultaneous capture of the phosphorylated substrate by the PhosphoSensor Acceptor coated with a Lewis Metal Chelate and the streptavidin SA Donor beads Upon laser excitation of the Donor beads the proximity of the Donor and PhosphoSensor Acceptor beads will generate an AlphaScreen signal between 520 and 620 nm In the absence of phosphorylation no signal should be observed Since the Acceptor beads allow for the detection of phos phates the activity of phosphatases can also be monitored us ing these beads Biotinylate
10. can be achieved by including either a glycine stretch or a LC long chain spacer N hydroxysuccinimidyl ester NHS or maleimide driven chemical reaction can be used for the addition of biotin to amino acid sequences The NHS driven reaction will target the secondary amine present on lysine residues and at the N terminal of the peptide On the other hand the maleimide driven reaction will target the sulphydryl group present in cysteine residues If the peptide contains many internal lysine residues which will be targeted by NHS it is recommended to have biotin integrated during peptide synthesis with the required spacer 2 Protein substrate Protein substrates can be biotinylated using either NHS or maleimide driven reactions in the same way as a peptide substrate see above However in order to prevent addition of biotin near the phosphorylation site of the substrate the presence of lysine or cysteine resi dues near the phosphorylation site should be evaluated to guide the use of one of the two chemistries NHS or maleimide driven reaction B Deciding on assay format 1 All in one well assay The all in one well homogenous assay format repre sents the format of choice for screening purposes In this format the kinase reaction is performed in the same well as the detection reaction Figure 2 For a detailed protocol description see section VI In a microplate add Kinase reaction Enzyme Inhibitor Biotinyla
11. ction reaction may alleviate the non specific bind ing of the substrate see section IV D Thus we do not use plus or minus ATP as an indication of the assay window D Evaluating optimal enzymatic assay conditions The following section presents the steps necessary to de termine the optimal ATP substrate and enzyme concentra tions in order to obtain an optimal signal window The opti mal signal window is defined here as the optimum S B ratio measured between the background signal basal signal obtained in the presence of staurosporine see note below and the maximal enzymatic activity Note 3 The non specific binding of some biotinylated peptides will be strongly diminished by the presence in the detection reaction of ATP and or some enzymes that are themselves phos phorylated for activation In these cases con trol incubations lacking either ATP or enzyme as a reference for background could be mis leading when evaluating assay window S B values For such sticky peptides it is advis able to determine assay window using control incubations containing all the reaction compo nents i e enzyme substrate and ATP in the presence or absence of a generic protein kinase inhibitor such as staurosporine Note 4 We do not recommend terminating the kinase reaction using EDTA at this stage of assay de velopment Since EDTA interferes to some ex tent with the detection by the PhosphoSensor Acceptor beads it should be
12. d substrate Peptide or protein ATP kinase ADP Emission Excitation 520 620 nm 680 nm Ue Streptavidin Biotinylated PhosphoSensor Donor Beads Phospho substrate Acceptor Beads Figure 1 J lustration of the detection of a phosphorylated peptide using the AlphaS creen PhosphoSensor Acceptor beads Legend LM Lewis Metal Chelate IV ASSAY DEVELOPMENT The AlphaScreen technology has been widely used for the de velopment of kinase assays using specific antibodies It is im portant to stress that the optimal detection buffer as well as ATP substrate and enzyme concentrations which have been determined for an antibody based AlphaScreen assay will not necessarily apply to an antibody free assay using the Phos phoSensor beads Consequently it is strongly advised to follow the assay development steps presented in this section A Deciding on substrate configuration The following guidelines should be followed when preparing a novel biotinylated substrate to be included in a kinase assay reaction monitored by the AlphaScreen technology The same substrate configuration applies for both an anti body based and an antibody free kinase assay develop ment using the AlphaScreen technology 1 Peptide substrate Peptide substrates should be designed such that they possess at least 20 carbons between the biotin label and the amino acid targeted for phosphorylation tyrosine serine or threonine For small peptides this
13. in triplicates to the wells of an Optiplate 384 microplate e 5 uL of kinase reaction dilution step d e 10 uL of Acceptor beads dilution step e e 10 uL of Donor beads dilution step f 28 h Incubate in the dark for 1 hour at room temperature i Read on an AlphaScreen reader j Incubate overnight in the dark and read again C AlphaScreen beads quality control 1 Prepare 1X control buffer dilute 1 5 mL of 10X control buffer with 13 35 mL of H2O and add 0 15 mL Tween 20 10 2 Prepare serial dilutions of the kit s positive control at 5yM Table VI Table VI Kit s positive control probe dilution using a peptide stock at 5 umol L Di final in Volume of 1X control ilu intermediate na tion assay mol L dilution buffer mol L uL uL 1X107 5 0 X 107 oO 180 umol L 3X10 1 5 X 10 60 of dil 1 1X10 5 0 X 10 60 of dil 2 3X 10 1 5X 10 60 of dil 3 1X10 5 0 X 10 60 of dil 4 ee 3x10 1 5 X 10 60 of dil 5 1X10 5 0 X 10 60 of dil 6 is 8 3x10 1 5 X 10 60 of dil 7 1X10 5 0 X10 60 of dil 8 ps Se 00 0 20 3 Dilute the PhosphoSensor Acceptor beads 1 100 in the 1X control buffer 4 Dilute the Streptavidin Donor beads 1 100 in 1X control buffer 29 VII 5 Protocol of addition The assay is performed in triplicates in a white Opti Plate 384 in a total volume of 25 uL a Add 5 uL of control phosphopeptide b Add 10 uL of PhosphoSensor Acceptor beads dilution 20 pg
14. ing optimal enzymatic assay conditions Termination of the kinase reaction Kinetics of the detection reaction Titration of PhosphoSensor Acceptor beads SUMMARY OF ASSAY DEVELOPMENT DETAILED PROTOCOLS Titration of phosphorylated versus non phosphorylated substrate All in one well and transfer kinase assays AlphaScreen beads quality control TROUBLESHOOTING GUIDE 24 24 29 30 BEFORE STARTING Receiving the AlphaScreen PhosphoSensor Kit Upon receiving the AlphaScreen PhosphoSensor Kit ensure that it is on blue ice and that the ice packs are not completely melted Verify that all components are present in the kit using the table below Provided Reagents and Materials The following kit sizes are available 1 000 assay points catalog number 6760307D 10 000 assay points catalog number 6760307M 50 000 assay points catalog number 6760307R The number of assay points is based on the use of 500 ng of each bead per well The reagents and materials provided in the AlphaScreen Phos phoSensor Kit are listed in the Table Table I Reagents and materials supplied 10 000 50 000 KIT assay assay COMPONENTS points points 6760307D 6760307M 6760307R 0 1 mL 1 0 mL i i 5 mg mL 5 mg mL pH 7 0 0 05 Proclin 300 Streptavidin Donor beads Stored in 100 mM Tris HCI a ja 2 p an pH 7 4 0 05 Proclin 300 g g Positive control bio LCK P Stored in 25 mM Hepes pH ye TEAN 7 4 0 05 Proclin 300 10X Co
15. mL final concentration c Add 10 uL of Streptavidin Donor beads dilution 20 ug mL final concentration d Incubate in the dark for 1 hour at room temperature e Read on an AlphaScreen reader h Incubate overnight in the dark and read again Note 10 The 10X detection buffer included in the kit may not be suitable for the detection of all phosphorylated peptides and should only be used for quality control of the beads Expected results maximum signal should be reached at 30 nM of control probe with an ECs between 1 5 to 15 nM The absolute maximum counts generated will be depend ent on the instrument used for the readout TROUBLESHOOTING GUIDE The following section describes the possible problems which could be encountered when developing an antibody free kinase assay using the AlphaScreen Technology If more information is required please consult your local PerkinElmer technical support division see page 34 for customer support informa tion 30 Possible Cause Effect Remedy No signal Detection Interference of EDTA with the conditions assay since EDTA is used to chelate the Mg ions titrate MgCl2 to limit the concentration of EDTA added for chelation Add a small volume of EDTA before adding the larger volumes of beads Stop the kinase reaction using a generic inhibitor such as staurosporine 1 10 uM ATP interferes with the detection 1 Increase the concentration of Acceptor beads to increase the tolerance
16. nteracting together Avoid premixing the Acceptor and Donor beads since signal will decrease substantially following 15 minutes of pre incubation Day to day Inappropriate standard operation variability procedures ensure that experimental procedures are the same from day to day 1 prepare the beads in the same area 2 ensure that incubation times are constant and temperature does not fluctuate in the room 33 MANUFACTURED BY PerkinElmer BioSignal Inc 1744 William Street Montreal Quebec Canada H3J 1R4 For further technical information or to place an order call PerkinElmer LAS Inc 710 Bridgeport Avenue Shelton CT 06484 USA 800 762 4000 or 203 925 4600 techsupport perkinelmer com European Headquarters PerkinElmer LAS Inc Imperiastraat 8 BE 1930 Zaventem Belgium Techsupport europe perkinelmer com Country Telephone Austria 0800 293 515 Belgium 0800 94 540 Denmark 80 88 3477 France 0800 90 77 62 Germany 0800 1 81 00 32 Italy 0800 79 03 10 Netherlands 0800 02 23 042 Norway 800 11 947 Spain 900 973 255 Sweden 020 79 07 35 Switzerland 0800 55 50 27 United Kingdom 0800 89 60 46 34 35 j gt PerkinElmer precisely PerkinElmer Life and Analytical Sciences Inc 710 Bridgeport Avenue Shelton CT 06484 4794 USA 800 762 4000 or 1 203 925 4602 www perkinelmer com For a complete listing of our global offices visit www perkinelmer com lasoffices M 6760307 01 36
17. ntrol buffer 100 mM MES pH 6 0 1M NaCl 0 05 Proclin 300 1 5 mL 1 5 mL 10X 10X Note before use e For maximum recovery of content briefly centrifuge the vials prior to removing the caps and resuspend the beads by vortexing e Reagents should be stored at 2 8 C e Acceptor and Donor beads should not be frozen and should be stored protected from light e 10X buffer may not be suitable as a kinase assay or as a detection buffer Recommended Additional Reagents and Materials Table II Recommended reagents and materials eee Suggested source Catalog Biotinylated N A N A substrate of choice HPLC water or Fisher Scientific W5 4 equivalent Sigma Aldrich Co A 3377 MgClz Sigma Aldrich Co M 9272 EDTA GIBCO 15575 038 OptiPlate 384 6007290 pack of 50 white opaque 6007299 pack of 200 384 well microplate TopSeal A Adhesive PerkinElmer Inc 6005185 sening pima Tween 20 Pierce 28320 Single channel N A N A penos o A A Sigma Aldrich Co S 4400 For lower volume additions 2 10 uL we recommend a pipet tor precision lt 2 For higher volume additions 25 1000 UL a pipettor precision of lt 1 is recommended Assay must be read using an AlphaScreen compatible reader such as all PerkinElmer EnVision multilabel plate readers with AlphaScreen module Fusion Alpha multilabel readers or AlphaQuest original AlphaScreen readers INTENDED USE The Alph
18. ossible e g 6 uL to allow for dilution of potential interferences during the de tection reaction Termination of kinase activity Before adding the detection beads it is recommended to terminate the kinase reaction by the addition of EDTA diluted in the optimal detection buffer For the concentra tion to use in the assay see section IV E If the signal window is too low using EDTA it is recom mended to use a generic inhibitor such as staurosporine to terminate the enzymatic reaction A titration should be per formed to determine the optimal staurosporine concentra tion Detection reaction Beads are added following the inactivation of the kinase It is recommended to use an overnight detection to increase the signal window This will not affect the pharmacological parameters of the assay since the enzyme has been inacti vated before the final detection step For detection buffer composition see section IV C Note 8 Acceptor beads can be titrated to increase tol erance to ATP See Section IV G 26 1 Prepare reaction buffer as suggested by the provider of the enzyme 2 Prepare detection buffer 10 mM Tris pH 7 0 100 mM NaCl 0 1 Tween 20 3 Protocol of addition for the all in one well assay The assay is performed in triplicates in a white Opti Plate 384 in a total volume of 28 UL a b Add 2 uL of enzyme diluted in the kinase reaction buffer Add 2 uL of inhibitor or buffer diluted in the
19. ours overnight incubation Figure 4B After 1 hour of incubation it was observed that substrate concentrations higher than 0 3 uM did not improve the signal window A decrease of the sig nal window was observed for ATP concentrations higher than 3 uM due to ATP interference Although similar results were obtained following an incubation period of 17 hours a greatly improved signal window was observed Thus for both incubation periods tested the optimal signal window was obtained when using 0 3 uM of biotinylated substrate and 3 uM of ATP Un der these conditions S B values of approximately 6 and 25 were observed following 1 hour and 17 hours detection time respectively A E 30000 biotin kemptide uM g 250004 0 20000 cg 1 5 3 150004 mS Hol 10000 4 2 lt 50004 TT o Uims 0 3 10 30 ATP uM B ore biotin kemptide uM 1750004 0 a 150000 S z 1250004 5 3 1000004 m BL 75000 lt amp 50000 i 25000 4 012 a 0 3 10 30 ATP uM Figure 4 Scheme of the preliminary kinase assay optimization Kinase reaction was performed in kinase reaction buffer 25 mM Hepes pH 7 4 100 mM NaCl 2 5 mM MgCl 1 mM DTT and 0 01 Tween 20 and beads were added in detection buffer 100 mM Tris pH 7 0 100 mM NaCl and 0 1 Tween 20 Incubations 25 uL were conducted in 384 well microplates Detection time was A 1hour and B 17 hours
20. re biotin non phospho and biotin phospho sub strate by making serial dilutions in kinase reaction buffer supplemented with ATP and EDTA Note 7 Supplementation with ATP and EDTA is neces sary to mimic closely the conditions that will be observed in an actual kinase assay Table IV shows an example of peptide dilution using a pep tide stock at 50 uM 24 Table IV Example of peptide dilution using a peptide stock at 50 umol L final in intermediate Volume of Kinase reac assay dilution tion buffer mow molt ul uL e axm 15x10 eoorais 1mo e exor 15x10 ooar mo o ixo 50x10 eoorais 120 mede eo ee ee ee 4 Dilute the PhosphoSensor Acceptor beads 1 100 in the detection buffer 5 Dilute the Streptavidin Donor beads 1 100 in the detec tion buffer 6 Protocol of addition The assay is performed in triplicates in a white Opti Plate 384 in a total volume of 25 uL a Add 5 uL of the biotinylated peptide dilution b Add 10 uL of PhosphoSensor Acceptor beads dilution 20 g mL final concentration c Add 10 uL of Streptavidin Donor beads dilution 20 pg mL final concentration d Incubate in the dark for 1 hour at room temperature e Read on an AlphaScreen reader 25 B All in one well and transfer kinase assays These assays are divided into three major steps Kinase reaction It is recommended but not always necessary to perform the kinase reaction in the smallest volume p
21. t buffer conditions to reduce non specific binding by varying e pH 6 to 8 e NaCl from 0 to 400 mM e Tween 20 from 0 to 0 1 and by evaluating Tris HCI HEPES or MES as potential buffers Kinase assay Ensure that the signal to background reagents ratio is established in the presence and absence of staurosporine since the presence of ATP and or enzyme in the reaction could alleviate the substrate non specific binding to the PhosphoSensor Acceptor beads 32 Possible Cause Effect Remedy High Kinase assay When using a full length kinase as a background reagents substrate ensure that it is not signal activated by phosphorylation Only use non activated kinase as a substrate High degree Microplates Warped or distorted microplates of signal avoid storage of microplate under variability heavy objects or next to sources of heat Uneven plate molding Light penetrating edges of microplate ensure use of black cover plate during bead incubation Incubate microplate in dark environment such as inside a drawer or cover microplate entirely with foil or material impenetrable to light Poorly fitted plate seals inducing evaporation of reaction mixture Instrument Temperature control problem within the instrument for the EnVision readers using the 1 07 software version adjust the internal temperature of the instrument For other readers consult the technical service department Assay conditions Beads are i
22. ted substrate ATP mix 384 well plate Incubate at RT ZII Reaction termination EDTA Incubate at RT Detection reaction PhosphoSensor Acceptor beads Streptavidin Donor beads Incubate at 1 hour at RT Read with an AlphaScreen capable reader Incubate overnight and read again Figure 2 Scheme of the all in one well assay format 2 Transfer assay The advantage of the transfer assay format is to dilute potential interfering reagents that are present in the kinase reaction before adding the detection beads This format can also be used for screening purposes It is especially useful when e the enzyme is intrinsically highly phosphorylated and therefore interferes with the detection of the phosphorylated peptide by the PhosphoSensor Acceptor beads e the signal generated is low and high ATP concen trations are required e g over 100 uM A scheme of the transfer assay protocol is illustrated in Figure 3 for a detailed protocol description see section VI Kinase reaction In a 96 well microplate add e Biotinylated substrate Enzyme ATP Incubate at RT 96 well plate Add EDTA to stop the reaction Dilution of reaction mix Dilute the reaction mix 1 20 Detection reaction In a microplate add e Diluted mix e PhosphoSensor Acceptor beads e Streptavidin Donor beads 384 well plate Incubate 1 hour at RT Read with AlphaScreen capable reader Figure 3 Scheme of the transfer assay format 12 C Titra
23. ter incubation of the kinase reaction the mix is di luted in order to obtain final concentrations of bioti nylated substrate between 0 and 1 uM in the detection reaction The detection reaction using the Acceptor and Donor beads is then conducted as for the all in one well assay format see section VI B E Termination of the kinase reaction EDTA is a commonly used chelator for termination of kinase reactions However excessive concentrations of EDTA should be avoided when performing a detection us ing the PhosphoSensor Acceptor beads It is recommended to perform all kinase assay development using 2 5 mM MgCl and then to determine the optimal con centration of EDTA required to terminate the enzymatic reaction If necessary titration of MgClz2 can also be per formed before EDTA titration Figure 6 shows the effect of increasing concentrations of EDTA on the PKA kinase reaction In this assay EDTA was added either before starting the kinase reaction or 2 hours following the initiation of the reaction Detection times of 1 and 17 hours were compared Figure 6A B To confirm the specificity of the reaction each incubation was performed in the presence or absence of a generic kinase inhibitor 10 uM staurosporine data not shown It was determined that 5 mM EDTA is sufficient to completely stop the kinase reaction while leaving an acceptable signal window S B ratio of 13 Overnight incubation of the detection reaction clearly res
24. tion of biotinylated phosphopeptide and or non phosphorylated peptide substrate To ease the optimization of an antibody free kinase assay using the AlphaScreen PhosphoSensor Acceptor beads we recommend obtaining the phosphorylated version of the peptide substrate Titration of this positive control in parallel with the biotinylated substrate will allow evaluation of the expected signal window It should be noted however that the availability of a phosphorylated peptide is not essential to develop a kinase assay using the AlphaScreen Phos phoSensor Kit For peptide titration assays the biotinylated peptide as well as the phosphopeptide if available should be added to the plate diluted in the kinase reaction buffer whereas the beads both Acceptor and Donor should be added to the plate in the detection buffer For the majority of biotinylated peptides tested the stan dard detection buffer composition is 10 mM Tris HCl pH 7 0 100 mM NaCl 0 1 Tween 20 However optimization of the detection buffer must be per formed when observing high non specific binding of the biotinylated substrate lonic strength nature of the buffer and pH of the detection reaction can influence the non specific binding of some peptide sequence Note 1 Do not use PBS since it contains phosphate which will bind to the PhosphoSensor Acceptor beads and displace the phosphorylated substrate Note 2 Be aware that the presence of ATP in the de te
25. ults in an improved S B ratio Figure 6B 500005 EDTA added at the beginning T 400004 EE EDTA added at the end 5 aS 30000 c 25 amp 8 20000 no amp 10000 i i 0 i ee Tot Saf a 0 0 2 5 5 0 12 5 25 0 EDTA mM B 14000005 EDTA added at the beginning 12000004 EEE EDTA added at the end te 1000000 c S E 8000004 fe 88 6000004 amp 400000 lt 2000004 i 0 E 0 0 2 5 5 0 12 5 25 0 EDTA mM Figure 6 Titration of EDTA Following the addition of EDTA a de tection time of 1 hour A or 17 hours B was used If no signal window is obtainable when using EDTA it is recommended to either use the transfer assay to dilute EDTA before reading or to add staurosporine at 1 10 uM to terminate the enzymatic reaction 19 Kinetics of the detection reaction Phosphopeptide detection using the PhosphoSensor Ac ceptor beads involves lower binding affinities than an anti body based detection method Thus following termination of the kinase activity it is recommended to incubate the detection reaction overnight to obtain the optimal signal window Figure 7 depicts the kinetics of the detection reaction fol lowing termination of PKA activity with 5 mM of EDTA A proportional increase in the signal window was observed as a function of time 1500000 e with staurosporine Se 1250000 o without staurosporine c D a 1000
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