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CY-1184

1. a y X User s Manual For Research Use Only Not for use in diagnostic procedures lt Example of Test Results Fig 1 Dose dependency of Catalytic Domain PKR aS 3 0 2 5 2 0 Z 1 5 1 0 0 5 0 0 GST PKR 252 551 ng Assay L AAO Fig 2 Km for ATP of Catalytic Domain PKR A 2 25 000 y 118 69x 415 07 R 0 9995 20 000 Km 3 5 uM 15 000 gt iral 10 000 S ra O O 5 000 F 4 0 0 25 50 75 100 125 150 175 200 Q ATP conc uM s amp 2 Raa CY 1184 17 Version 150807 Q AO v PKR EIF2AK2 Kinase Assay Kit O Pd a yCLex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 3 Effect of NaCl on the kinase activity of Catalytic Domain PKR S A450 0 50 100 150 200 250 300 350 400 450 500 NaCl mM 4 Fig 4 Effect of C16 a PKR inhibitor on the kinase Nity of Catalytic Domain PKR 100 90 80 70 E 3 60 2 50 2 2 40 Z 30 20 4 10 0 001 0 010 0 100 1 000 10 000 100 000 amp C16 uM Q Raa CY 1184 18 Version 150807 Q AO CLex Fig 5 Effect of poly I C and C16 on the kinase activity of Full Length PKR For Research Use Only Not for use in diagnostic procedures PKR EIF2AK2 Kinase Assay Kit User s Manual A450 0 8 0 6 0 4 0 2 0 0 200uM C16 100uM C16 E 50uM
2. B glycerophosphate ImM DTT 1x U Cocktail 2 Protein A Agarose Beads 50 bead sum Add 5 mL of 1X PBS to 1 5 g of proteings agarose beads Shake 2 hours at 4 C spin down Wash beads twice with PBS Resuspend begn 1 volume of PBS Can be stored for 2 weeks at 4 C 3 Poly L lysine coated Plate amp Coat the plate with 25 ug mL g y L lysine PLL in PBS for 4 12 h at 37 C Subsequent to a washing step with PBS y 2 Treatment of Cells W 1 Plate adherent cells S 6 well plate dish at 1 x 10 cells plate 2 Incubate the cul ish at 37 C for 12 16 hours in CO2 incubator 3 Change the medium to fresh media containing 10 ug mL poly I C See the section Materials Required bape Provided 4 neubau culture dish at 37 C for appropriate time Ad o Raa CY 1184 15 Version 150807 ee oo y A User s Manual For Research Use Only Not for use in diagnostic procedures v rg PKR EIF2AK2 Kinase Assay Kit O g gt 3 Preparing Cell Lysates N 1 Remove media and wash cells with ice cold PBS and aspirate mS A 2 Add 1 mL of ice cold Cell Lysis Buffer to each plate 6 well plate dish and shake at cao rpm on an orbital microplate shaker for 120 minutes each at 4 C Q 3 Scrape off and transfer the lysate to microcentrifuge tubes and nicroentituagds 000 rpm for 10 minutes at 4 C 4 Transfer the supernatant to a new tube The supernatant is the cell lysate ffecessary the lysate can be stored at 70
3. C wN 4 Immunoprecipitation and assay kinase activity D 1 Take 100 uL of the cell lysate and add anti human PKR poha antibody CycLex Co Ltd Cat CY P1043 1 2 ug and incubate with gentle rocking for ours at 4 C 2 Add 20 uL of Protein A Agarose Beads 50 bead slung incubate with gentle rocking for 1 3 hours at 4 C Go 3 Microcentrifuge for 30 seconds at 4 C Wash the Ain A agarose beads 3 times with 500 uL of Cell Lysis Buffer and once with Kinase Buffer K amp p on ice during the washes 4 Resuspend the protein A agarose beads wp 20 0 uL of Kinase Buffer and use 10 uL as an enzyme sample to measure PKR activit ygd ording to the procedure in the step 3 to 12 of 2 Standard Assay in the section TE gO eOn above Please take care to transfer the erin A agarose beads to the wells of the Antibody coated Microplate as little as possible q Note Although we suggest to c aguct experiments as outlined above the optimal experimental conditions will vary depende on the parameters being investigated and must be determined by the individual user Esp y an appropriate cell lysate must be optimized by titration of the cell lysate and setting t ount which shows OD value does not exceed plateau range in a dose response Curve WARRANTY OR GUARANTEE OF PERFORMANCE USING THESE PROC S IS MADE OR IMPLIED amp N Ad A Raa CY 1184 16 Version 150807 e oo y rag PKR EIF2AK2 Kinase Assay Kit E lt
4. C16 10uM C16 uM C16 e 0uM C16 ol m 15 20 20 30 poly I C ng mL d a lt Y N A450 o 30 ng mL polyI C 15 ng mL polyl C 7 5 ng mL polyI C 4 0 ng mL polyl C C16 uM 2 Raa CY 1184 19 Version 150807 Q AO wy PKR EIF2AK2 Kinase Assay Kit re gt 4 User s Manual A eg y For Research Use Only Not for use in diagnostic procedures Q Fig 6 Effect of poly I C on the kinase activity of Full Length PKR N 1 2 1 0 0 8 0 6 A450 0 4 0 2 0 0 0 1 1 0 10 0 100 0 1 000 0 poly I C ng mL vV Fig 7 Time course of poly I C effect on native PKR unoprecipitated from HepG2 cell Procedures as a guideline HepG2 cell extract LP ATP Plate HepG2 cells at a density of 0 5x10 O HepG2 cell extract I P ATP cells well in 6 well plate Culture at 37 C O N Add poly I C to final conc of 10 ug mL to each well Culture at 37 C for indicated times Remove the medium Gently wash the cells with ice cold PBS J Discard PBS Add 1 mL of Cell ILysis Buffer to each well Incubate at 4 C for 2 hour with gentle shaking Harvest the lysates 0 O a ee ee ere ee ee eee Centrifuge at 13 000 rpm for 5 minutes 30 40 50 60 7 0 at 4 C HepG2 cell exposed time to poly I C hr Use 100 u
5. Mi e PKR EIF2 Catalytic domain Availa Qi om CycLex Co Ltd PKR EIF2AK2 Human Active Cat CY SPP80 e PKR EIF2 Full length Availap from CycLex Co Ltd PKR EIF2AK2 Positive Control Full length Cat CY E1 184 1 aA e Optional BSA containigg Wlution buffer 20mM Hepes KOH pH 7 5 1 BSA ImM EDTA 2mM DTT 50mM NaClan 50 glycerol e Optional C16 10 up Mk N specific inhibitor of PKR Available from Sigma Cat I9785 Make 1 mM stock solution wit SO and dilute 1 100 with Kinase Buffer e Optional P I C Polyinosinic polycytidylic acid sodium salt A synthetic analog of double strand NA dsRNA Available from Sigma Cat P1530 e inhibitor cocktail Available from Cell Signaling Technology Inc Cat 58 pe es Anti PKR antibody Available from CycLex Co Ltd Cat CY P1043 A Raa CY 1184 5 Version 150807 Q gt A PKR EIF2AK2 Kinase Assay Kit v y 3X User s Manual For Research Use Only Not for use in diagnostic procedures Q Precautions and Recommendations 3 e Allow all the components to come to room temperature before use mS te e All microplate strips that are not immediately required should be returned to the zip lock Qa which must be carefully resealed to avoid moisture absorption O e Do not use kit components beyond the indicated kit expiration date S e Use only the microtiter wells provided with the kit Q e Rinse all detergent residue fro
6. A Solution One vial containing 2 mL of 0 5 yorra pH 8 0 Ready to use HRP conjugated Detection Antibody One b ontaining 12 mL of HRP horseradish peroxidase conjugated anti GST antibody Ready to use 4 Substrate Reagent One bottle conn mL of the chromogenic substrate tetra methylbenzidine TMB Ready to use Stop Solution One bottle containi mL of 1 N H2SO Ready to use Y ge NS X amp N Ad o Raa CY 1184 4 Version 150807 ee oe N gt Py PKR EIF2AK2 Kinase Assay Kit v y 3X User s Manual For Research Use Only Not for use in diagnostic procedures Q Materials Required but not Provided 3 e Orbital microplate shaker 7 Ss e Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips S e Wash bottle or multichannel dispenser for plate washing amp e Microcentrifuge and tubes for sample preparation 2 e Vortex mixer y RY e Microplate washer optional Manual washing is possible but not prefexgbTe e Plate reader capable of measuring absorbance in 96 well plates atdyal wavelengths of 450 nm 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be us The plate can also be read at a single wavelength of 450 nm which will give a somewhat higher Pading e Optional Software package facilitating data generation K 500 or 1000 mL graduated cylinder G e Reagent reservoirs Q e Deionized water of the highest quality wW e Disposable paper towels
7. L of the clear lysate as a sample for P kinase assay 2 Raa CY 1184 20 Version 150807 e oo N gt PKR EIF2AK2 Kinase Assay Kit O Cl e User s Manual y y J Ser S anua For Research Use Only Not for use in diagnostic procedures 4 References 3 1 Clemens MJ Elia A The double stranded RNA dependent protein kinase PKR str e and function J Interferon Cytokine Res 17 503 524 1997 RY 2 Kaufman RJ The double stranded RNA activated protein kinase PKR In Sone g N et al editors Translational Control of Gene Expression Cold Spring Harbor LaboraQy Press 2000 p503 528 2000 Yy 3 Robertson HD Mathews MB The regulation of the protein kinase PKR wy A Biochimie 78 909 914 1996 on S ee SS 4 Williams BR Signal integration via PKR Sci STKE RE2 2001 5 Stark GR Kerr IM Williams BRG Silverman RH and Schraigye RD How cells respond to interferons Annu Rev Biochem 67 227 264 1998 6 Williams BRG PKR a sentinel kinase for cellular stress O ene 18 6112 6120 1999 7 Judware R and Petryshyn R Mechanism of action of lular inhibitor of the dsRNA dependent protein kinase from 3T3 F442A cells J Biol Chem 2 21685 21690 2992 8 Kumar A Yang Y L Flati V Der S Kadereig9 Deb A Haque J Reis L Weissmann C and Williams B R Deficient cytokine sign in mouse embryo fibroblasts with a targeted deletion in the PKR gene role of IRF 1 and NESeappaB EMB
8. O J 16 406 416 1997 9 Kumar A Haque J Lacoste J Hiscot Wand Williams B R Double stranded RNA dependent protein kinase activates transcription faghNF KB by phosphorylating IkB Proc Natl Acad Sci U S A 91 6288 6292 1994 G N euzzi A Schafer R Kumar A Williams B R G Aguet signaling in mice devoid of double stranded RNA dependent 6106 1995 10 Yang Y Reis L F L Pavlovic M and Weissmann C Defici protein kinase EMBO J 14 11 Meurs E F Galabru QPrrber G N Katze M G and Hovanessian A G Tumor suppressor function of the interfegon mduced double stranded RNA activated protein kinase Proc Natl Acad Sci U S A 90 232 0 1993 12 Barber G N bach M Thompson S Jagus R and Katze M G Mutants of the RNA dependen tein kinase PKR lacking double stranded RNA binding domain I can act as transdominan yhibitors and induce malignant transformation Mol Cell Biol 15 3138 3146 1995 13 Takizawa Ohashi K and Nakanishi Y Possible involvement of double stranded RN A activated protein Qe in cell death by influenza virus infection J Virol 70 8128 8132 1996 14 Qh Rodriguez D Rodriguez J R and Esteban M The apoptosis pathway triggered by the interferon induced protein kinase PKR requires the third basic domain initiates upstream of Bcl 2 involves ICE like protease Virology 231 81 88 1997 ei Lee S B Rodriguez D Rodriguez J R
9. Y ra PKR EIF2AK2 Kinase Assay Kit G ycLex User s Manual re For Research Use Only Not for use in diagnostic procedures gt Non Radioisotopic Kit for Measuring PKR EIF2AK2 Activity CycLex PKR EIF2AK2 Kinase Assay Rit Cat CY 11 84 N 2 Intended CSG pisiveasecoucswersovdoovetenavacseereasiontevens 1 OT AS ee cscs E E l y JEOLKOLCIUTO NTO t TAAIE E 2 SY Principle of the Assay ccccceesseeeeeeeeeees 2 3 rw Materials Provided ccccccccssseseeeeceeeeenes 4 oO Materials Required but not Provided 5 Ni Precautions and Recommendations 6 oy Detailed Protocol ecececceceseeeseeeessessessees 7 13 Evaluation of Resullts ccecceecseeeeeeeees 14 4 Assay Characteristics cccccecssceseseeeeeees 14 Qy Troubleshooting ccccccccccesssseeeeeceeeeeeeeens 14 RY Reagent Stability ccccssseeeeeeeeeeeeens 14 Sample Preparation cccccseesseeceeeeeeeeeees 15 1 Example of Test Result eee 1X20 RETENC ee EE oe 22 Related Products ccccccccceecccceeeeeeeeees t 2 I sO ntended Use The CycLex Research Product Cyc KR EIF2AK2 Kinase Assay Kit is primarily designed to measure the activities of purified dependent protein kinase PKR or recombinant PKR for the rapid and sensitive evaluation of hibitors or activators The phospho serine specific monoclonal antibody used in this assay kit Hay been demonstrated to recognize the phosphorylated serine 51
10. and Esteban M The apoptosis pathway triggered by the Ke at CY 1184 21 Version 150807 ee oo Y Raa CY 1184 22 Version 150807 T Q y 3X User s Manual For Research Use Only Not for use in diagnostic procedures 4 interferon induced protein kinase PKR requires the third basic domain initiates upstream of amp and involves ICE like protease Virology 231 81 88 1997 16 Nakamura T Furuhashi M Li P Cao H Tuncman G Sonenberg N Gorgun CZ Hotami GS Double stranded RNA dependent protein kinase links pathogen sensing with stress an tabolic homeostasis Cell 140 338 48 2010 Q Related Products ee CycLex PKR EIF2AK2 Kinase Assay Kit Cat CY 1184 y PKR EIF2AK2 Human Active Cat CY SPP80 SY PKR EIF2AK2 Positive Control Full length Cat CY E1184 1 rw Anti Human PKR EIF2AK2 Polyclonal Antibody Cat CY P1043 Qy gt a h PRODUCED BY CycLex Co Ltd 1063 103 Terasawaph Ina Nagano 396 0 Japan Q Fax 81 265 769618 Crete products are supplied for research use only CycLex CircuLex products and com nts thereof may not be resold modified for resale or used to manufacture commercial pr s without prior written approval from CycLex Co Ltd To inquire about licensing for S commercial use please contact us via email N gt Py PKR EIF2AK2 Kinase Assay Kit
11. bate at 30 C for 60 minutes shaking at ca 300 on an orbital microplate shaker Yon Select the suitable Reaction Buffer Poly I C plus or Poly I C minus X type of sample and PKR Positive control See the 6 Prepare Reaction Buffer for kinase reaction a 1 Preparation of Working Solution above amp 5 Stop the kinase reaction by addition of 10 uL of ED amp A Solution to each well Mix well by pipetting gt 6 Transfer 100 uL of the reaction mixture to each weff of the Antibody coated Microplate cover with plate sealer or lid and incubate at room erature ca 25 C for 60 minutes shaking at ca 300 rpm on an orbital microplate shaker R 7 Wash five times by filling each well withefpdsh Buffer Remove residual Wash Buffer by gentle tapping or aspiration A 8 Pipette 100 uL of HRP conjugated Bftection Antibody to each well cover with plate sealer or lid and incubate at room temperatur a 25 C for 60 minutes shaking at ca 300 rpm on an orbital microplate shaker Q 9 Wash five times by filling cAr well with Wash Buffer Remove residual Wash Buffer by gentle tapping or aspiration O A 10 Add 100 uL of Substxate Reagent to each well and incubate at room temperature ca 25 C for 5 20 minutes shaki t ca 300 rpm on an orbital microplate shaker 11 Add 100 uL Or Solution to each well in the same order as the previously added Substrate Reagent 12 Measure rbance in each well using a spec
12. d must besdetermined by the individual user Especially an appropriate amount of the enzy ust be optimized by titration of the enzyme and setting the amount which show OD value does not exceed plateau range in dose response curve RS Q amp Q Raa CY 1184 13 Version 150807 ee oe N gt PKR EIF2AK2 Kinase Assay Kit O yclex User s Manual For Research Use Only Not for use in diagnostic procedures 4 Evaluation of Results c Average the absorbance values for the Positive Control and all experimental sample dupliggfe Values when applicable When Catalytic Domain PKR See the 5 Prepare Positive Contro of I Preparation of Working Solution in the section Detailed Protocol above is included in ng assay as an internal control for the kinase reaction the absorbance value should be greater gn 1 0 with a background less than 0 25 when using Reaction buffer Assay Characteristics G The CycLex Research Product CycLex PKR EIF2AK2 Kinase Assay Kit been shown to detect the activity of purified PKR and column fractions containing PKR The ass ay be used to follow the purification of PKR or can be used to detect PKR kinase activity in t Nimmunoprecipitate from cell lysates using anti PKR antibody Troubleshooting Ks 1 All samples and controls should be assayed in duplicate when a standard assay is being performed using the protocol described in the section Detailed focol Incuba
13. est compounds co Sv it 1S necessary to conduct the control experiment of Solvent Control Assay at least ye every experiment and Inhibitor Control Assay at least once for the first experiment in a glition to Test Compound Assay as indicated in the following table When test compounds c an inhibitory effect on PKR activity the level of Test Compound Assay weaken as com with Solvent Control Assay 8 Inhibitor screening using Catalytic Domain PKR y Test Compound Inhibitor Control Assay Reagents Assay ARAY Assay Reaction Buffer Poly I C minus 80 uL Se 80 uL Test Compound 10 uL Solvent of Test Compound C16 10 uM 10 uL l k Catalytic Domain PKR 10uL 10 uL 10 uL See the 6 Prepare Reaction Buffers for kinase reaction 2 9 1 Preparation of Working Solution above See the section Materials Required but not Provided Save 20 ng uL of Catalytic Domain PKR See the sectf6n Materials Required but not Provided and 5 Prepare Positive Control s in 1 Preparation of Workig Sptution above 1 Following the table above add the re ts to each well of the Non coated Microplate Finally initiate reaction by adding 10 uL o alytic Domain PKR to each well and mixing thoroughly at room temperature Cover with g sealer or lid and incubate at 30 C for 60 minutes shakin at ca 300 rpm on an orbital mic te shaker 2 Follow the step 5 to 12 of Standard A
14. hour at Toa anti phospho Q eIF2a serine 51 Cast monoclonal Wash the wells aS antibody O O 9 Add 100 f HRP conjugated anti GST antibody Q Incubate for 1 hour at room temp Wash the wells t Add 100 uL of Substrate Reagent Incubate for 5 to 20 minutes at room temp Add 100 uL of Stop Solution t Measure absorbance at 450 nm Ke at CY 1184 3 Version 150807 ee oo y y 3X User s Manual For Research Use Only Not for use in diagnostic procedures 4 Materials Provided c All samples and standards should be assayed in duplicate The following components are sigue and are sufficient for the one 96 well microplate kit RY Non coated Microplate for kinase reaction One microplate supplied ready to use withy96 wells 12 strips of 8 wells in a clear zip lock bag Antibody coated Microplate One microplate supplied ready to use with 96 well pS strips of 8 wells in a foil zip lock bag with a desiccant pack Wells are coated with anti plagsp o eIF2a serine 51 monoclonal antibody as a capture antibody 10X Wash Buffer One bottle containing 100 mL of 10X buffer containing Ween 20 Kinase Buffer One bottle containing 20 mL of 1X buffer used amp Reaction Buffer and sample dilution amp 20X ATP One vial of lyophilized ATP Naz salt L 20X DTT Four vials of lyophilized dithiothreitol g 10X GST elF 2a Substrate One vial containing 37 5 ne lyophilized recombinant GST elF2a EDT
15. in Eukaryotic Initiation Factor IF2a which is efficiently phosphorylated by PKR Additionally column fractions of a cells cell lines or tissues can be assayed for PKR activity with the CycLex Research ProducteCyc ex PKR EIF2AK2 Kinase Assay Kit if the appropriate dose of PKR specific inhibitor e g Clas used Applications of this Kude 1 Screening inhibit rs or activators of PKR EIF2 AK2 2 Evaluating t V sttects of pharmacological agents on PKR EIF2AK2 activity in vitro 3 Monitoring purification of the kinase activity of PKR EIF2AK2 This assay Kits for research use only and not for use in diagnostic or therapeutic procedures e WBon receipt store all components at 4 C n t expose reagents to excessive light N a Raa CY 1184 1 Version 150807 Q PKR EIF2AK2 Kinase Assay Kit yelex User s Manual For Research Use Only Not for use in diagnostic procedures 4 N Q O Q Introduction 3 The double stranded RNA activated protein kinase PKR also known as eukaryotic RMeation initiation factor 2 alpha kinase 2 EIF2AK2 is a ubiquitously expressed serine threonine pretein kinase that plays a key role in the innate immunity response to viral infection in higher eukaryoteseayid has also been implicated in several cellular signal transduction pathways 1 4 The dsRNAs mediated activation leads to autophosphorylation of PKR and allo phosphorylate its natural substrate the a subunit of initiation fact
16. itor ATP Minus Sif No Enzyme Assay Reagents Control Control Control Assay Reaction Buffer Poly I C plus or minus Reaction Buffer ATP Poly I C minus 10 uL native PKR sample PKR Positive Control 10 a Buffer of native PKR sample Select the suitable Reaction Buffer Poly I C plus or E minus for the type of PKR Positive control See the 6 Prepare Reaction Buffers for kinase reaction in Preparation of Working Solutions above See the 6 Prepare Reaction Buffers for kinase Ooo in 1 Preparation of Working Solutions above See the section Materials Required but not P Sea above KK 20 ng uL of Catalytic Domain PKR or Raitsu of Full Length PKR See the section Materials Required but not Provided and 5 Prepare Pos ontras in 1 Preparation of Working Solution above 1 Following the table above add thA reagents to each well of the Non coated Microplate Finally initiate reaction by adding 10 of your native PKR sample or PKR Positive Control or Buffer of native PKR sample to e ell and mixing thoroughly at room temperature Cover with plate sealer or lid and incub t 30 C for 60 minutes shaking at ca 300 rpm on an orbital microplate shaker NN 2 Follow the step 5 to of 2 Standard Assay above Note Although Dusest to conduct experiments as outlined in the table above the optimal experim conditions will vary depending on the parameters being investigated an
17. m glassware e Use deionized water of the highest quality e Do not mix reagents from different kits Ci e The buffers and reagents in this kit may contain preservatives Kher chemicals Care should be taken to avoid direct contact with these reagents lt Q Do not mouth pipette or ingest any of the reagents 7 gt e Do not smoke eat or drink when performing the pay or in areas where samples or reagents are handled SS e Dispose of tetra methylbenzidine TMB contig is solutions in compliance with local regulations e Avoid contact with the acidic Stop Solutiog ghd Substrate Solution which contains hydrogen peroxide e Wear gloves and eye protection when ling immunodiagnostic materials and samples of rat origin and these reagents In case of contact With the Stop Solution and the Substrate Solution wash skin thoroughly with water and seek mes attention when necessary e Biological samples may be 00 aminated with infectious agents Do not ingest expose to open wounds or breathe acres ear protective gloves and dispose of biological samples properly handli p Solution P amp A 2 Raa CY 1184 6 Version 150807 e CAUTION Sulfuric N d is a strong acid Wear disposable gloves and eye protection when o gt Py PKR EIF2AK2 Kinase Assay Kit N gt PKR EIF2AK2 Kinase Assay Kit O Cl e User s Manual y y J Ser S anua For Research Use Only Not for use in diag
18. mL Stes t e solution in small aliquots e g 100 uL at 20 C 5 Prepare PKR Positive Control s A 6 Catalytic Domain PKR amp Ist step Dilute the PKR EIR2 Catalytic domain not provided See the section Materials Required but not yovided above with BSA containing dilution buffer not provided See ipDection Materials Required but not Provided above to be 100 ng uL AN 2nd step Dilute fst step enzyme solution 1 5 with Kinase Buffer Make sure a final concentgaddgn of the PKR EIF2 Catalytic domain must be 20 ng uL Full Length PKR S Dilute PK F2 Full length not provided See the section Materials Required but not Provided e with Kinase Buffer to be 1 units uL RS Q Ad o Raa CY 1184 T Version 150807 o PKR EIF2AK2 Kinase Assay Kit y 3X User s Manual A For Research Use Only Not for use in diagnostic procedures Q 6 Prepare Reaction Buffers for kinase reaction N Reaction Buffer Poly I C minus ac Ww For assays with Catalytic Domain PKR prepare Reaction Buffer Poly I C minys gt Quantity required 90 uL assay Mix following components Discard any unused the buffer use Kinase Buffer 10X GST eIF2a Substrate Solution 20X DTT Solution 20X ATP Solution Reaction Buffer Poly I C plus S For assays with Full Length PKR or native PKR sample Sa Reaction Buffer Poly I C plus Quantity required 90 uL assay Mix following co
19. mpepyents Discard any unused the buffer after use 7 Kinase Buffer 10X GST elF2 a Substrate Solution xO 20X DTT Solution 20X ATP Solution Pz Sug mL poly I C 10 02mL 1 002uL 100 2 uL F Not provided See the seeff n Materials Required but not Provided above Reaction Buffer ATP Pa minus For assays of ER activity prepare Reaction Buffer ATP Poly I C minus Quantity required 90 PLNA 1x following components Discard any unused the buffer after use Componefits 96 assays 10 assays assay eke Kinase Buffer 10X eIF2a Substrate Solution gto Solution ne ae 1000 100 Rs Raa CY 1184 8 Version 150807 Q gt PKR EIF2AK2 Kinase Assay Kit O Cl e User s Manual y y J Ser S anua For Research Use Only Not for use in diagnostic procedures s S 2 Standard Assay N 1 Remove the appropriate number of Non coated Microplate and Antibody coated plate wells from the pouch and place them into the well holder Return any unused wells to th pouch refold seal with tape and store at 4 C 2 Prepare all samples diluted with Kinase Buffer as needed amp 3 Add 10 uL of sample and PKR Positive Control See the 5 Prepare PKR reff Control s in 1 Preparation of Working Solution above to each well of Non coated Mici plate on ice 4 Start the kinase reaction by addition of 90 uL of Reaction Buffer wer well cover with plate sealer or lid and incu
20. nly Not for use in diagnostic procedures 4 Sample Preparation 3 Numerous extraction and purification methods can be used to isolate PKR The followinggestocols have been shown to work with a number of different cells and enzyme sources and are ered as examples of suitable methods Crude samples can frequently be used without dilutio ile more concentrated or highly purified PKR should be diluted It is strongly advised that tee user always perform an initial experiment to determine the proper dilution to be used in subse experiments This need not be any more than a single time point assay using serial dilutions of t ude extract cell lysate or sample fraction taken prior to a purification step One eight well strip e plate should be sufficient for this initial experiment All sample preparation should be performed at 4 C and recovered fractions should be kept at 4 C to prevent loss of enzymatic activity lt CAUTION It should be noted that this assay kit detects not only BRR activity but also other protein kinases in crude extracts and column samples Mh presence of PKR protein in the samples should be traced by other methods e g pestern blotting Immunoprecipitation Protocol Followed by anar ai Activity 1 Preparation of Solution and Reagent A O 1 Cell Lysis Buffer lt 20 mM Tris HCI pH 7 5 250 mM NaCl 10 glyce l 0 5 Nonidet P 40 1 mM EDTA 1 mM EGTA 0 2 mM PMSF 1 ug mL pepstatin 0 5 L leupeptin 5 mM NaF 2 mM Na3VOa 2 mM
21. nostic procedures 4 Detailed Protocol The CycLex PKR EIF2AK2 Kinase Assay Kit is provided with removable strips of w o the assay can be carried out on separate occasions using only the number of strips required for 7 determination Since conditions may vary running an aliquot of the appropriate PKR Posi amp Ve Control should be included in each assay Disposable pipette tips and reagent troughs should used for all transfers to avoid cross contamination of reagents or samples amp 1 Preparation of Working Solutions amp w 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 1 ash Buffer to 900 mL of deionized distilled water ddH20O Mix well Store at 4 C for two s or 20 C for long term storage A Ci 2 Prepare 20X ATP Solution by adding 1 6 mL of ddH20 to the vjadkof 20X ATP lyophilized Mix gently until dissolved The final concentration of the 20X ATP S6lution should be 1 25 mM Store the solution in small aliquots e g 200 uL at 20 C 3 Prepare 20X DTT Solution by adding 0 25 mL of ddH20 te the vial of 20X DTT lyophilized Mix gently until dissolved The final concentration of the 7 gt Solution should be 200 mM Store the solution in small aliquots e g 50 uL at 20 C S 4 Prepare 10X GST eIF2a Substrate Solution by pers 0 6 mL of ddH20 to the vial of GST eIF2a Substrate lyophilized Mix gently until ite The final concentration of the 10X GST eIF2a Substrate Solution should be 62 5 pg
22. or eIF2 resulting im tepid inhibition of translation and suppression of virus spread 5 6 PKR also has been implicat regulating other cellular functions such as differentiation 7 transcription 8 9 signal transduction 10 cell growth 11 12 and apoptosis in the event of virus infection and other forms of cellular sett 13 15 It was reported that the activation of PKR in adipose and liver tissue is rsa by obesity 16 In the absence of PKR metabolic deterioration due to excess energy or nutritien 18 alleviated These findings demonstrate that PKR is an important component of inflammation comp that responds to nutrients and organelle dysfunction lt the kinase to Principle of the Assay rs The CycLex Research Product CycLex ee Assay Kit is a semi quantitative immunoassay for the kinase activity of PKR This produ n be used to determine the presence of the kinase activity in purification column fractions or to folloW the kinetics of a purified or partially purified PKR protein as well as screening PKR inhibitors or a ators The protocol for the quantitative measurement kinase activity involves incubation of the PKR sample with its substrate GST eIF2a fusion protein in the presence of Mg and ATP followed by transfer this kinase reaction mixture to the of microtiter plate which is pre coated with a monoclonal antibody specific for phospho el20 S5 1 for trapping only phosphorylated substrate The amount of phosphorylated subst
23. rate on ell is measured by a horseradish peroxidase coupled anti GST antibody which then c es the conversion of the chromogenic substrate tetra methylbenzidine TMB from ac ess solution to a blue solution or yellow after the addition of stopping reagent The color is quantged by spectrophotometry and reflects the relative amount of the kinase activity in the sample For kinetic analysis the sampl ontaining PKR is added to the wells in a similar fashion and at varying times the reaction is ed by the addition of a chelator sodium ethylenediaminetetraacetate EDTA and the amount of phorylated substrate determined as mentioned before The CycLex Research Product CycLex PKR EIF2AK2 Kinase Assay Kit is designed to accurately determine the presence an amount of PKR activity in purification column fractions and to determine non istopieg he ic analysis of the kinase activity of PKR Careful attention to extraction methods and the assOy otocol will provide the investigator with a reliable tool for the evaluation of the kinase activity O 0 O amp gt A 2 Raa CY 1184 2 Version 150807 o v PKR EIF2AK2 Kinase Assay Kit O Pa a ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Summary of Procedure S Incubate for 1 hour at 30 C Add of 10 uL of EDTA Solution to stop kinase ragon S gt Transfer 100 uL of reaction mixtures t QF antibody costed wells Ci Incubate for 1
24. ssay above Note Although we sung conduct experiments as outlined in the table above the optimal experimental ca ions will vary depending on the parameters being investigated and must be determi ed by the individual user Especially an appropriate amount of the enzyme mus optimized by titration of the enzyme and setting the amount which shows OD e does not exceed plateau range in dose response curve amp O Q Q amp Q Raa CY 1184 11 Version 150807 Q N P PKR EIF2AK2 Kinase Assay Kit a U GA y 3X ser s Manual A For Research Use Only Not for use in diagnostic procedures Q Inhibitor screening using Full Length PKR or native PKR samples Oo Asay Reagens Test Compound Solvent Control mee COMtrol Assay Reagents Assay Assay Reaction Buffer Poly I C plus 80 uL 80 uL 7 Test Compound Solvent of Test Compound C16 10 uM Full Gengi PKR 10 uL 10 uL 10 uL See the 6 Prepare Reaction Buffers for kinase reaction in 1 Preparation of ving Solution above See the section Materials Required but not Provided above 1 units uL of Full Length PKR See the section Materials Required b Provided and 5 Prepare Positive Control s in 1 Preparation of Working Solution above Yom 1 Following the table above add the reagents to each well re Non coated Microplate Finally initiate reaction by adding 10 uL of Full Length PKR dor your na
25. tion times or temperatures significantly different from those specified may give e ous results 2 The reaction curve is nearly a straight line if the ki Ges of the assay is of the first order Variations in the protocol can lead to non linearity of the cur Scan assay kinetics of other than first order For a non linear curve point to point or quadratic cope methods should be used 3 Poor duplicates accompanied by elevated in for wells containing no sample indicate insufficient washing If all instructions in the Detayg Protoco were followed accurately such results indicate a need for washer maintenance Q 4 Overall low signal may indicate thepglesiccation of the plate has occurred between the final wash and addition of Substrate Reagent D pot allow the plate to dry out Add Substrate Reagent immediately after wash O Reagent Stabilit g All of the reagents 1 d in the CycLex Research Product CycLex PKR EIF2AK2 Kinase Assay Kit have been teste tability Reagents should not be used beyond the stated expiration date Upon receipt kit reagents Id be stored at 4 C Antibody coated Microplate should be stored in the original foil bag sealed by by zip lock and containing a desiccant pack For research i only not for use in diagnostic or therapeutic procedures Q Raa CY 1184 14 Version 150807 ee oe N gt PKR EIF2AK2 Kinase Assay Kit O Cl e User s Manual y y J Ser S anua For Research Use O
26. tive PKR sample to each well and mixing thoroughly at room temperature Cover wi yplate sealer or lid and incubate at 30 C for 60 minutes shaking at ca 300 rpm on an obialafropl shaker 2 Follow the step 5 to 12 of 2 Standard Assay ove Note Although we suggest to conduct exp ents as outlined in the table above the optimal experimental conditions will vary nding on the parameters being investigated and must be determined by the indiyidual user Especially an appropriate amount of the enzyme must be optimized b ation of the enzyme and setting the amount which shows OD value does not exegrplateau range in dose response curve amp Raa CY 1184 12 Version 150807 Q PKR EIF2AK2 Kinase Assay Kit Cycle Users Ma G2 y J ser s Manua For Research Use Only Not for use in diagnostic procedures II Special considerations for measuring precise PKR activity O In order to measure the activity of PKR correctly it is necessary to conduct the control expefrment of Inhibitor Control Assay at least once for every experiment and ATP Minus Control y at least once for the first experiment in addition to No Enzyme Control Assay as indi in the following table The level of A450 increases in Test Sample Assay when the sample ains PKR enzyme activity although the high levels of A450 are not observed in Inhibitor ge Assay ATP Minus Control Assay and No Enzyme Control Assay Inhib
27. trophotometric plate reader at dual wavelengths of Dual wavelengths of 450 550 or 450 595 nm can also be used Read the plate at 450 a single wavelength can be used Wells must be read within 30 minutes of adding the 2 Raa CY 1184 9 Version 150807 o N 4 PKR EIF2AK2 Kinase Assay Kit A User lt aa ycLex ser s Manual For Research Use Only Not for use in diagnostic procedures Q 9 Oy Note 1 Complete removal of liquid at each step is essential to good performance After the last wash remove any remaining Wash Buffer by aspirating or decanting Invert the plate a Nolot 1 against clean paper towels Wh 9 Note 2 Reliable signals are obtained when either O D values do not exceed 0 25 units for the blank no enzyme control or 2 5 units for Positive Controls N Note 3 If the microplate reader is not capable of reading absorbance greater t he absorbance of Positive Controls perform a second reading at 405 nm A new O D yas measured at 405 nm is used to determine PKR activity of off scale samples The r gs at 405 nm should not replace the on scale readings at 450 nm amp Raa CY 1184 10 Version 150807 Q AO PKR EIF2AK2 Kinase Assay Kit gt Cl e User s Manual y y J Ser S anua For Research Use Only Not for use in diagnostic procedures 3 Recommendations N I Special considerations for screening inhibitors In order to estimate the inhibitory effect on PKR activity in your t

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