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CY-1175

1. 0 2 4 6 8 10 PKC m units 100 ul reaction w Fig 2 Typical time course of kinase reaction with ng rat brain A 1 8 1 6 14 i2 B 1 0 os 0 6 04 Q o Q o 0 15 30 45 oe Reaction Time min S o e o OX C E Q o S 4 PKC Super Family Kinase Assay Kit o oO clex User s Manual 4 v y For Research Use Only Not for use in diagnostic procedures 8 Fig 3 Effect of PKC inhibitor H 9 on activity of rat brain Protein kinase C 3 AS 120 S 110 Qo E 100 S 90 Y S go O s 70 Y 60 amp so 5 3 40 a 30 D 20 10 0 0 1 1 0 10 0 100 0 1000 0 H9 uM o Fig 4 RESOURCE Q column elution profile of PK eon rabbit brain extract xO CaCD Phosphatidylserine 2 mM EGTA NaCl 1 6 1000 1 4 1 2 750 1 0 z 2 X 0 8 500 g 0 6 z 0 4 250 v Q w 0 Qe O 10 2 30 40 50 60 Fr No o e Q qu CY 1175 17 Versions 151015 Q amp C 4 PKC Super Family Kinase Assay Kit e LA cle User s Manual A e 2X y For Research Use Only Not for use in diagnostic procedures 8 Fig 5 Detectable activities of PKC isozymes using CycLex PKC Super Family Kinase Assay Kit 3 cPKC Ww PKC a TER KS PKC fI TH Q PKC II amp PKC y 4444 nPKC c PKC 6 n PKC amp PKC yn S PKC 0 PKC u N A aPKC S P
2. 1 Grow cells on 10 cm dish at 80 confluent overnight n 2 Treat the cells with desired drug or compound for appropriate time 3 Scrape the cells with a rubber policeman after wash the three times with cold PBS 4 Resuspend the cell pellet with 500 uL of an ap riate extraction buffer for example 20 mM Tris HCl pH 7 5 75 mM NaCl 5 glycerol aet Triton X100 20 mM f glycerophosphate 1 mM EDTA 1 mM EGTA 0 2 mM PMSF X amp ug mL pepstatin 0 5 ug mL leupeptin 5 mM B glycerophosphate 2 mM NaF 1 mM Nao 5 Sonicate the resuspended cells on ice for die seconds using 10 15 second pulses 6 Centrifuge at 100 000 x g for 1 hour uec 7 Aliquot cleared cell lysate to a n microfuge tube These samples are now ready for analysis according to the instructions proggde d in the Detailed Protocol Store the cell lysate at below 70 C Q NOTE THE ABOVE PROCEDURES ARE INTENDED ONLY AS A GUIDELINE THE OPTIMAL EXP ENTAL CONDITIONS WILL VARY DEPENDING ON THE PARAMETERS ING INVESTIGATED AND MUST BE DETERMINED BY THE INDIVIDUALNWSER NO WARRANTY OR GUARANTEE OF PERFORMANCE USING T ROCEDURES IS MADE OR IMPLIED amp o S a KS Q qu CY 1175 15 Versions 151015 LS d 4 PKC Super Family Kinase Assay Kit e LA cle User s Manual A e 2X y For Research Use Only Not for use in diagnostic procedures Example of Test Results 9 Fig 1 Typical dose dependency curve of PKC from rat brain C 3 0 amp
3. 8 Ohno S Akita Y Konno Y et al A novel phorbo Mo nid ren kinase nPKC distantly related to the protein kinase C family Cell 53 731 741 I 9 Osada S Mizuno K Saido TC et al A phor Mester receptor protein kinase nPKC a new member of the protein kinase C family predominant ressed in lung and skin J Biol Chem 265 22434 22440 1990 nA 10 Osada S Mizuno K Saido TC gt A new member of the protein kinase C family nPKC 0 predominantly expressed in selgnusct Mol Cell Biol 12 3930 3938 1992 11 Ono Y Fujii T Ogita K et al ein kinase C zeta subspecies from rat brain its structure expression and properties Proc Nagy Sci USA 86 3099 3103 1989 12 Selbie LA Schmitz Pei C Sheng Y et al Molecular cloning and characterization of PKC iota an atypical isoform otein kinase C derived from insulin secreting cells J Biol Chem 268 24296 2430 3 13 Nishizuka Y Infyacellular signaling by hydrolysis of phospholipids and activation of protein kinase C Science 258 66 614 1992 14 Kerane Qu Dutil EM Newton AC Protein kinase C is regulated in vivo by three functionally disting Ph sphorylations Curr Biol 12 1394 1403 1995 15 Newttn A Protein kinase C structure function and regulation J Biol Chem 270 28495 28498 1995 Ka JW Keranen LM Newton AC Reversible exposure of the pseudosubstrate domain of protein Q kinase C by phosphatidylserine and diacylglycerol J Biol Chem 267 15263 15266 1992 LN at CY 1175 19 Versions 151015
4. and allowed to phosphorylate the bound substrate in th presence of Mg and ATP The amount of phosphorylated substrate is measured by bindj it with a AK IFII a anti phospho CPI 17 threonine38 monoclonal antibody followed nding with horseradish peroxidase conjugated anti mouse IgG which then catalyzes the c rsion of the chromogenic substrate tetra methylbenzidine TMB from a colorless solution to a ye solution or yellow after the addition of stopping reagent The color is quantified by spectro metry and reflects the relative amount of PKC activity in the sample For kinetic analysis the s e containing PKC is added to the wells in a similar fashion and at varying times the reaction i pped by the addition of a chelator sodium ethylenediaminetetraacetate EDTA and the amount gp osphorylated substrate determined as before The CycLex Research Product CycLex PKC Supe ad ay Kinase Assay Kit is designed to determine non isotopic kinetic analysis of PKC Careful MD to extraction and purification methods and the assay protocol will provide the investigatorQyith a reliable tool for the evaluation of PKC activity o qu CY 1175 3 Version 151015 LS d PKC Super Family Kinase Assay Kit o LA p j y y O e Vclex User s Manual 4 For Research Use Only Not for use in diagnostic procedures 8 Summary of Procedure 3 Add 100 uL of sample to the wells S 4 Incubate for 30 min at 30 C Wash the wells g Add 100 uL of Anti phospho CPI 17 T38 mon
5. KCG PKC 1 4 t e Alpha o Beta I BD 4 Beta II BID e Gamma y e Delta Epsilon s Zeta C Eta n e Theta 0 1 Iota 1 A450 Enzyme conc ng 100 ul Reaction q e Soa hj amp n S PKC Super Family Kinase Assay Kit e 4 Vclex User s Manual For Research Use Only Not for use in diagnostic procedures amp References 9 Nishizuka Y Intracellular signaling by hydrolysis of phospholipids and activation of proteinase C Science 258 607 614 1992 S 2 Nishizuka Y Protein kinase C and lipid signaling for sustained cellular responses FASRBY 9 484 496 1995 3 Parker PJ Coussens L Totty N et al The complete primary structure of proteinggmase C the major phorbol ester receptor Science 233 853 859 1986 CY 4 Coussens L Parker PJ Rhee L et al Multiple distinct forms of bovine 2 ads protein kinase C suggest diversity in cellular signaling pathways Science 233 859 866 Ist 5 Knopf JL Lee MH Sultzman LA et al Cloning and expression of ope protein kinase C cDNAs Cell 46 491 502 1986 TN 6 Ohno S Kawasaki H Imajoh S et al Tissue specific expressio three distinct types of rabbit protein kinase C Nature 325 161 166 1987 lt 7 Ono Y Fujii T Ogita K et al The structure expressio Gd properties of additional members of the protein kinase C family J Biol Chem 263 6927 6932 aS
6. L of anti phospho CPI 17 T38 monoclonal antibody AK 1F1 1 Ready to use HRP conjugated Anti mouse IgG One vNontaining 12 mL of HRP horseradish peroxidase conjugated anti mouse IgG Ready to use Q Substrate Reagent One bottle contain 20 mL of the chromogenic substrate tetra methylbenzidine TMB Ready to use Stop Solution One bottle suppliegyfady to use containing 20 mL of 1 N H2SO Ready to use amp eo qu CY 1175 5 Versions 151015 LS d n S PKC Super Family Kinase Assay Kit e 4 e Vclex User s Manual For Research Use Only Not for use in diagnostic procedures amp Jf dp o aterials Required but not Provided PKC Positive Control S 1 Native PKC from rat brain is available from Promega Cat V5261 NN 2 Recombinant PKC isoenzymes are available from CycLex Recombinant PKC iso mes may lose or decrease dependency on Ca2 and or phospholipid Q CycLex s recombinant PKC amp PKC alpha Xenopus Active Cat CY SPP61 q PKC beta I Human Active Cat CY SPP62 Q PKC beta II Human Active Cat CY SPP63 n PKC delta Human Active Cat CY SPP64 Yon PKC epsilon Human Active Cat CY SPP65 QN PKC gamma Human Active Cat CY SPP66 a PKC eta Human Active Cat CY SPP67 oO PKC iota Human Active Cat CY SPP68 m PKC mu Human Active Cat CY SPP72 amp PKC nu Human Active Cat CY SPP73 Yon PKC zeta Human Active Cat CY SPP75 amp PKC I
7. LS d S PKC Super Family Kinase Assay Kit e 4 v Vclex User s Manual For Research Use Only Not for use in diagnostic procedures 17 Mochly Rosen D Localization of protein kinases by anchoring proteins a theme in nal transduction Science 268 247 251 1995 QN Ww vw 18 Nishizuka Y Protein kinase C and lipid signaling for sustained cellular responses FASEBRS 484 496 1995 o Related Products ES Recombinant PKC isoenzymes w PKC alpha Xenopus Active Cat CY SPP61 Yon PKC beta I Human Active Cat CY SPP62 C PKC beta II Human Active Cat CY SPP63 NX PKC delta Human Active Cat CY SPP64 Yon PKC epsilon Human Active Cat CY SPP65 m PKC gamma Human Active Cat CY SPP66 amp PKC eta Human Active Cat CY SPP67 oO PKC iota Human Active Cat CY SPP68 amp PKC mu Human Active Cat CY SPP72 4 PKC nu Human Active Cat CY SPP73 Q PKC zeta Human Active Cat CY SPP75 KG Antibody Q Anti Phospho CPI 17 Thr38 Monoclonal Antiljdv Clone AK 1F11 Cat CY M1024 S e e G y q NS PRODUCED BY RS CycLex Co Ltd Q 1063 103 Terasawa Ina Nagano 396 0002 Japan Fax 81 265 48 7618 ircuLex products are supplied for research use only CycLex CircuLex products and ents thereof may not be resold modified for resale or used to manufacture commercial cts without prior written approval from CycLex Co Ltd To inquire about licensing for comme
8. PKC Super Family Kinase Assay Kit 4 v Vclex User s Manual For Research Use Only Not for use in diagnostic procedures gt Non Radioisotopic Kit for Measuring PKC Activity 3 CycLex PKC Super Family Kinase AssagKit Cat CY 1175 o Intended Use seee 1 spl 1 e Introduction eeeeeeeeeeer 2 3 QN Principle of the Assay penna 3 4 a Materials Provided usine rre 5 Yon Materials Required but not Provided 6 S Precautions and Recommendations 7 qe Detailed Protocol ssusse 8 12 amp Evaluation of Results ssss 13 amp Assay Char cteristies uere een estre tae trip 13 Q Troubleshootite s scccccasssscnsaccssasssansaabvaavvede 13 o Reagent SEIIIIDY sienten tta ep eiu 13 gt Sample Preparation esses erar rtt tiu 14 Example of Test Results 1 References ccecceceseceeceeeeseeessseeseeeeseees S Related Products uss 0 C Intended Use The CycLex Research Product CycL x KC Super Family Kinase Assay Kit is primarily designed to measure the activities of purified Qiptein kinase C PKC for the rapid and sensitive evaluation of inhibitors or activators The phosj M9 specific monoclonal antibody used in this assay kit has been demonstrated to recognize t hospho threonine38 residue in CPI 17 which is efficiently phosphorylated by PKC Ad ally column frac
9. cessary onduct the control experiment of Solvent control at least once for every experiment and Inhibitor ES considerations when screening activators and inhibitors e at CY 1175 10 Versions 151015 LS d Vclex n PKC Super Family Kinase Assay Kit User s Manual For Research Use Only Not for use in diagnostic procedures G S oO Q amp control at least once for the first experiment in addition to Test sample as indicated in the foll ig table When test chemicals cause an inhibitory effect on PKC activity the level of A450 is weak as compared with Solvent control The high level of A450 is not observed in Inhibitor controK ssually A450 0 2 S Solvent nhibitor control S control Assay reagents Test sample Kinase Reaction Buffer Ca PS plus 80 uL 80 up 80 uL 10X Inhibitor or equivalent 10 uL z Solvent for Inhibitor AE 2 PKC Inhibitor X 10 uL PKC Positive Control N 10 pL 10 pL 10 pL or Enzyme Sample H 3 H H Kinase Reaction Buffer Ca PS plus See the section Preparation of Workin Nation above PKC Inhibitor e g 10X H 9 100 uM See the section Materials a not Provided above PKC Positive Control See the section Materials Required but not Pro above 1 Following the above table add the Reagents to each wellgpf the microplate Finally initiate reaction by adding 10 uL of PKC Positive Control or your Enzyme Sampl
10. e to each well and mixing thoroughly at room temperature Cover with plate seal cubate at 30 C for 30 minutes 2 Follow steps 5 12 in the Standard Assay above Ww Special considerations when measuring preci Kc activity In order to measure the activity of PKC co Sry it is necessary to conduct the control experiment of Inhibitor control at least once for every riment and Ca PS minus control at least once for the first experiment in addition to No MN control as indicated in the following table Although the level of A450 increases in Test sampleg en PKC enzyme activity is in the sample the high level of A450 is not observed in Inhibitor co AM ATP minus control and No enzyme control eo Ca PS No Assay reagents ut puru minus eed enzyme control control Kinase Reaction ami vg plus 80 uL 80 uL 80 uL 80 uL Kinase Reaction Bu a PS minus 80 uL PKC Inhibitor y 10 uL Kinase Buffer vided 10 uL 10 uL 10 uL 10 uL Buffer for YodHenzyme fraction 10 pL PKC Posi Control 10 pL Fnzgp mpl 10 pL 10 uL 10 pL M eis Buffer Ca PS plus and Ca PS minus See the section Preparation of Working Solution above p nhibitor e g 10X H 9 100 uM See the section Materials Required but not Provided above ui Positive Control Native PKC is recommended to see dependency on Ca2 and phospholipid See the section aterials Requ
11. e Reagent Do not allow the plate to dry out Add Substrate Reagent immediately after wash o Rea tabilit All or reagents included in the CycLex Research Product CycLex PKC Super Family Kinase Assa have been tested for stability Reagents should not be used beyond the stated expiration date Up ceipt kit reagents should be stored at 4 C except the ATP must be stored at 20 C Coated a plates should be stored in the original foil bag sealed by the zip lock and containing a desiccant qu CY 1175 13 Versions 151015 Q PKC Super Family Kinase Assay Kit 4 v Vclex User s Manual For Research Use Only Not for use in diagnostic procedures amp n C Q Sample Preparation 9 Numerous extraction and purification methods can be used to isolate PKC The following g tocols have been shown to work with rat cerebellum as enzyme sources and are provided as ples of suitable methods Concentrated or highly purified PKC should be diluted It is strongly a d that the user always perform an initial experiment to determine the proper dilution to be sedan subsequent experiments This need not be any more than a single time point assay using serial dil s of the crude extract cell lysate or sample fraction taken prior to a purification step One ei ell strip of the substrate plate should be sufficient for this initial experiment All sample ation should be performed at 4 C and recovered fractions should be kept at 4 C to prevent loss O
12. iled Protocol 9 The CycLex Research Product CycLex PKC Super Family Kinase Assay Kit is pro A with removable strips of wells so the assay can be carried out on separate occasions using only tha imber of strips required for the particular determination Since experimental conditions may vary aliquot of PKC Positive Control should be included in each assay Disposable pipette tips and Q3gent troughs should be used for all liquid transfers to avoid cross contamination of reagents or samp Y Preparation of Working Solution Q 900 mL of deionized distilled water ddH2O Mix well Store at 4 two weeks or 20 C for long term storage 1 Prepare a working solution of Wash Buffer by adding 100 mL of the xis Buffer provided to gt vial of 20X ATP provided e 20X ATP Solution should be 2 Prepare 20X ATP Solution by adding 1 6 mL of ddH20 to lyophilized Mix gently until dissolved The Final concentratio 1 25 mM Store the solution in small aliquots e g 100 uL at 3 Agitate 10X Phosphatidylserine PS vigorously by soniggion or vortex for a minute prior to use on ice o 4 Prepare Kinase Reaction Buffer Ca PS plus by mixi following reagents voas 10 assays 1 assay Kinase Buffer provided G5 mL 830 uL 83 uL 20X ATP Solution 5 mL 50 uL 5 uL 50X CaCh provided d 0 2 mL 20 uL 2uL 10X PS provided 1 0 mL 100 uL 10 uL Total 10 mL 1000uL 100 pL You will need 80 90 f Kinase Reactio
13. ired but not Provided above Q qu CY 1175 11 Versions 151015 LS d n g le e Vclex User s Manual rey For Research Use Only Not for use in diagnostic procedures amp 1 Following the above table add the Reagents to each well of the microplate Finally initia e reaction by adding 10 uL of PKC Positive Control or your Enzyme Sample or Buffer ch well and mixing thoroughly at room temperature Cover with plate sealer Incubate at 30 Cor 30 minutes S 2 Follow steps 5 12 in the Standard Assay above e e Q qu CY 1175 12 Versions 151015 Q amp C d n S PKC Super Family Kinase Assay Kit e 4 cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures amp Evaluation of Results S 1 Average the absorbance values in duplicates of PKC Positive Control and all experimental tmples when applicable When PKC Positive Control is included as an internal cont for the phosphorylation reaction the absorbance value should be greater than 1 0 with a backgr less than 0 2 Q 2 For screening of purification chromatography fractions on graph paper plot t ean absorbance values for each of the samples on the Y axis versus the fraction number on the s to determine the location of the eluted purified PKC R 3 For kinetic analysis on graph paper plot the mean absorbance values f h of the time points on the Y axis versus the time of each reaction minutes on the X a
14. kenzymatic activity CAUSION It should be noted that this assay kit detects not only PRC activity but also other protein kinase activities e g Protein kinase A activated by partial proteolysis Rho kinase and ILK in crude extract and column sam n the absence of Ca and phospholipid You should trace PKC protein level Ay western blotting in column fractions amp Preparation of rat or rabbit brain extract amp amp 1 Homogenize fresh 10 15 g of rat or rabbit brain in four v es of ice cold Extraction Buffer 20 mM Tris HCl pH 7 5 0 25 M sucrose 5 mM EDTA 5 GTA 20 mM p mercaptoethanol 1 mM PMSF 1 ug mL pepstatin 0 5 ug mL leupeptin 5 B glycerophosphate 2 mM NaF 2 mM Na3VOs in a Potter Elvehjem tissue homogenizo o 2 Centrifuge the homogenate for 20 min at 2g x g to pellet the insoluble membrane organelle fraction Lt 3 The supernatant was centrifuged at rong g for 1 hr Column Purification of nenia PKC 4 Load the resultant high speed su Life Sciences or equivalent buffer pH 7 5 containing ug mL pepstatin 0 5 u g atant onto a 2 x 8 cm column of DEAE Sephacel GE Healthcare n exchange resin equilibrated with Buffer A 20 mM Tris HC1 EGTA 1 mM EDTA 10 mM p mercaptoethanol 0 5 mM PMSF 1 leupeptin 5 mM B glycerophosphate 2 mM NaF and 2 mM Na3VOs 5 Wash the column with column volumes of Buffer A 6 Sequentially su protein with a 0 0 3 M KC1 gradient in Buffer A Pool the fractions con
15. n Buffer Ca PS plus per assay well Mix well Discard any unuse ase Reaction Buffer Ca PS plus after use 5 When measuring precise Re activity see below prepare Kinase Reaction Buffer Ca PS minus by mixing following re ts amp 96 assays 10 assays 1 assay Kise Buffer provided 8 3 mL 830 uL 83 pL 2X ATP Solution 0 5 mL 50 uL 5uL X EGTA provided 0 2 mL 20 uL 2uL gme 1 0 mL 100 uL 10 uL A Total 10 mL 1000uL 100 pL In the case of assaying individual column fractions we recommend you to measure the e kinase activity in the absence of Calcium PS as well as in the presence of these in parallel PKC Super Family Kinase Assay Kit 4 v Vclex User s Manual For Research Use Only Not for use in diagnostic procedures amp S Standard Assay 3 Remove the appropriate number of microtiter wells from the foil pouch and place them inge well holder Return any unused wells to the foil pouch refold seal with tape and store at 4 Ctwe 2 Prepare all samples diluted with Kinase Buffer as needed All samples shoulqgye assayed in duplicate w To assay individual column fractions add 10 uL of each fraction to the well Phe assay plate on ice Duplicate wells containing 10 uL of PKC Positive Control should be igel ed in each assay as a positive control for phosphorylation Y Kinase Reaction Buffer Ca PS minus per well cover with plate r and incubate at 30 C for 4 Begin
16. nce and relative amount of PKC activity in purification column fractions and foe non isotopic kinetic analysis of PKC activity Careful attention to extraction methods and the assay protocol will provide the investigator with a reliable tool for the evaluation of PKC ycLex PKC Super Family Kinase Assay Kit can be used for measuring most of recombinant Pelle isozymes including all cPKCs as well as aPKCs and several nPKC e g PKC 6 e and 0 see page qu CY 1175 2 Versions 151015 LS d PKC Super Family Kinase Assay Kit 4 e Vclex User s Manual For Research Use Only Not for use in diagnostic procedures amp n C Q Principle of the Assay 9 The CycLex Research Product CycLex PKC Super Family Kinase Assay Kit is a sifgte site semi quantitative immunoassay for PKC activity Plates are pre coated with a substrate corregBending to recombinant CPI 17 Protein kinase C potentiated inhibitor protein of 17kDa MEN contains threonine38 residues that can be efficiently phosphorylated by PKC The detector antibody is AK IF11 an antibody that specifically detects only the phosphorylated CPI 17 threonine38 The Lex Research Product CycLex PKC Super Family Kinase Assay Kit might be used to determig the presence of PKC activity in purification column fractions or to follow the kinetics of a punfigd PKC as well as screening PKC inhibitor or activator To perform the test the sample is didyted in Kinase Buffer pipetted into the wells
17. nhibitor e g 10X H 9 100 uM available from adn Cat 13312 10 mM stock solution DMSO diluted 1 100 in Kinase Buffer 3 Pipettors 2 20 uL 20 200 uL and 200 1000 uL pregjon pipettors with disposable tips Precision repeating pipettor b gt Wash bottle or multichannel dispenser forghate washing Microcentrifuge and tubes for sample i Plate reader capable of measuring absorbance in 96 well plates at dual wavelengths of 450 nm 540 nm Dual wavelengths of 450 55 450 595 nm can also be used The plate can also be read at a single wavelength of 450 nm wg will give a somewhat higher reading 500 or 1000 mL graduates inder A Reagent reservoirs A Deionized water finest quality Disposable papqpjowels E S a qu CY 1175 6 Version 151015 LS d n S PKC Super Family Kinase Assay Kit e 4 v Vclex User s Manual For Research Use Only Not for use in diagnostic procedures amp Precautions and Recommendations 9 Store PKC Positive Controls at below 70 C and the ATP at 20 C in aliquots Store other components at 4 C Do not expose reagents to excessive light Avoid freeze thaw cycles S Allow all the components to come to room temperature before use Q All microplate strips that are not immediately required should be returned to the anf pouch which must be carefully resealed to avoid moisture absorption Q Do not use kit components beyond the indicated kit expiration da
18. oclonal antibagh AKI 11 1 Incubate for 30 min at room ie Wash n wells gt Add 100 uL of HRP conjugated anti mouse IgG 9 i Incubate for 30 mi oom temp Wash the wells amp e Add 100 uL of Substrate Reagent KG o Add 100 uL of Stop south o Measure absorbance at 50 nm eo Q qu CY 1175 4 Versions 151015 Q amp C PKC Super Family Kinase Assay Kit 4 e Vclex User s Manual For Research Use Only Not for use in diagnostic procedures amp n C Q Materials Provided 9 All samples and standards should be assayed in duplicate The following components are ua and are sufficient for the one 96 well microtiter plate kit S Microplate One microplate supplied ready to use with 96 wells 12 strips of 8 wells in yfoil zip lock bag with a desiccant pack Wells are coated with recombinant CPI 17 as a substrate amp 10X Wash Buffer One 100 mL bottle of 10X buffer containing Tween 20 c Kinase Buffer One bottle containing 20 mL of 1X buffer used for Kinase Reon Buffer and sample dilution v 10X Phosphatidylserine PS One vial containing 1 2 mL of 500 u g miffsphatidylserine 50X CaCl One vial containing 0 4 mL of 125 mM CaCb used Kinase Reaction Buffer Ca PS plus 50X EGTA One vial containing 0 4 mL of 100 mM EGTAgused for Kinase Reaction Buffer Ca PS minus Q 20X ATP One vial of lyophilized ATP Naz salt m o Anti Phospho CPI 17 T38 Monoclonal Antibod e vial containing 12 m
19. rcial use please contact us via email LN att CY 1175 20 Version 151015 LS d
20. s No liable 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 the PKC positive control Nof s If the microplate reader is not capable of reading absorbance greater than the absorbance of the Weel positive control perform a second reading at 405 nm A new O D values measured at dV 405 nm is used to determine PKC activity of off scale samples The readings at 405 nm should not replace the on scale readings at 450 nm LN at CY 1175 9 Version 151015 LS d PKC Super Family Kinase Assay Kit 4 v ycLex User s Manual Kinetic Assay 1 w Begin kinase reaction by addition of 90 uL of Kinase Reaction B For Research Use Only Not for use in diagnostic procedures Remove the appropriate number of microtiter wells from the foil pouch and place them inge well holder Return any unused wells to the foil pouch refold seal with tape and store at 4 Cte Prepare all enzyme samples diluted with Kinase Buffer as needed All enzyme sarep es should be assayed in duplicate To assay enzyme sample add 10 uL of each enzyme sample to the vag the assay plate Duplicate wells containing 10 uL of PKC Positive Control should be incid in each assay as a positive control for phosphorylation Ca PS plus in duplicate per well in timed intervals suggested interval is 1 minutes but sh be individually determined for each system After the final addition inc
21. taining y high kinase activity and concentrate by ultrafiltration to 1 5 original volume and adjust the KCI concentration M 7 Load the pP eluate kinase rich fraction to a phenyl Sepharose column 1 X 5 cm equilibrated with B 20 mM Tris HC1 buffer pH 7 5 containing 0 5 mM EGTA 0 5 mM EDTA 10 mM p m A Wem 0 5 mM PMSF 1 ug mL pepstatin 0 5 ug mL leupeptin 5 mM B gl erophosphate 2 mM NaF and 2 mM Nas VO and 10 glycerol containing 1 5 M KCl 8 Wash the column with Buffer B containing 1 5 M KCl until the absorbance at 280 nm reached seline Q qu CY 1175 14 Versions 151015 LS d 4 v Vclex User s Manual For Research Use Only Not for use in diagnostic procedures n S PKC Super Family Kinase Assay Kit e amp 9 Elute PKC with a gradient of 1 5 0 M KCI in Buffer B Pool the fractions containing high Y activity and concentrate by ultrafiltration to approximately 2 ml 10 Dialyzed the pooled kinase fraction against Buffer C 20 mM KPOs pH 7 5 0 5 mM A 0 5 mM EGTA 1 mM DTT and 5 mM f glycerophosphate 2 mM NaF and 2 mM Na3V and 10 glycerol for 2 hr at 4 C 7 11 Load the phenyl Sepharose column eluate onto a hydroxylapatite column 2 ml Livrarea Buffer C O 12 Washed the column with buffer C until the absorbance at 280 nm reached b ne 13 Elute PKC with a gradient of 0 02 0 28 M KPO in Buffer C at 0 5 cw collecting 1 ml fractions 4 Preparation of cell lysate mA gt
22. te oO Use only the microtiter wells provided with the kit Rinse all detergent residue from glassware RG Use deionized water of the highest quality qe Do not mix reagents from different kits S The buffers and reagents in this kit may contain preserv toes or other chemicals Care should be taken to avoid direct contact with these reagents Ss Do not mouth pipette or ingest any of the reagenten Do not smoke eat or drink when performi e assay or in areas where samples or reagents are handled amp Dispose of tetra methylbenzidine TMB taining solutions in compliance with local regulations Avoid contact with Substrate Solty Which contains hydrogen peroxide Avoid contact with Stop Solutionggfich contains Sulfuric Acid Q n case of contact with the BP Solution and the Substrate Solution wash skin thoroughly with water and seek medical attention en necessary Biological samples ON contaminated with infectious agents Do not ingest expose to open wounds or breath osols Wear protective gloves and dispose of biological samples properly CAUTION Sujlfuric Acid is a strong acid Wear disposable gloves and eye protection when h ndling Stop Solution S a KS Q qu CY 1175 7 Versions 151015 o d Q qu CY 1175 8 Versionft 151015 Q d n S PKC Super Family Kinase Assay Kit e 4 v ycLex User s Manual For Research Use Only Not for use in diagnostic procedures amp Deta
23. the kinase reaction by addition of 90 uL of Kinase i piae P Ne Ca PS plus and or 30 minutes m 5 Wash wells five times with Wash Buffer making sure ec is filled completely Remove residual Wash Buffer by gentle tapping or aspiration 6 Pipette 100 uL of Anti Phospho CPI 17 T38 Monoclopal Antibody into each well cover with plate sealer or lid and incubate at room temperate ca 25 C for 30 minutes Discard any unused antibody after use y 7 Wash wells five times as same as in step 5 lt 8 Pipette 100 uL of HRP conjugated Anti se IgG into each well cover with plate sealer or lid and incubate at room temperature ca 2 for 30 minutes Discard any unused conjugate after use Q 9 Wash wells five times as same as in 5 10 Add 100 uL of Substrate Regn to each well and incubate at room temperature ca 25 C for 5 15 minutes Y 11 Add 100 uL of Stop Yim to each well in the same order as the previously added Substrate Reagent NN 12 Measure absorbar each well using a spectrophotometric plate reader at dual wavelengths of 450 540 nm D avelengths of 450 550 or 450 595 nm can also be used Read the plate at 450 nm if only a e wavelength can be used Wells must be read within 30 minutes of adding the Stop Solutiday Note 1 Comfllete removal of liquid at each step is essential to good performance After the last wash Q any remaining Wash Buffer by aspirating or decanting Invert the plate and blot it st clean paper towel
24. tions of cultured primary cell cell line or tissue homogenate can be assayed PKC activity with the CycLex Research Product CycLex PKC Super Family Kinase Assay Kit Athe appropriate dose of PKC inhibitor e g H 9 is used Applications of this ki rie 1 Monitoring thefulyfication of PKC 2 Screening inhibitors or activators of PKC 3 Detecting t ects of pharmacological agents on PKC activity This assay kityS for research use only and not for use in diagnostic or therapeutic procedures g receipt store all components at 4 C 19h t expose reagents to excessive light Q qu CY 1175 1 Versions 151015 LS c PKC Super Family Kinase Assay Kit 4 v Vclex User s Manual For Research Use Only Not for use in diagnostic procedures amp n C Q Introduction 9 Members of the protein kinase C PKC family of serine threonine protein kinases Re been implicated in numerous cellular responses in a large variety of cell types PKC Dm N in multiple biochemical processes relevant to cell growth differentiation and transformatsdg and play critical roles in transducing signals from a plethora of extracellular receptors incluging those for hormones neurotransmitters growth factors and antigens 1 2 At present the PKC family of serine threonine specific protein kinases incl eleven known members that are expressed in a variety of tissues and cell types Based on simil in primary amino acid sequence and domain struct
25. tribution intracellular localization and cofactor requirements suggesting that they gre independently regulated through response to discrete ligands and that they may possess disti A icici substrates 18 It is possible that the specific combination of isoenzymes expressed in Mgiven cell determines the outcome of the PKC dependent response in that particular cell e Measurement of PKC activity The protocol generally rea as most sensitive for the quantitative measurement of PKC activity involves incubation of the P mple with substrate either a natural or synthetic polypeptide such as a histone III S in the presgnc of Mg and P labeled ATP The reaction is terminated by spotting a sample onto a phosphocgfiflose P81 filter paper disc followed by washing extensively to remove unincorporated radiola d the incorporated radioactivity on P81 filter is counted While sensitive this method is laborg sive generates hazardous radioactive waste and depends on a radioisotope of short half life It is cularly unsuitable when kinase assays are only performed on an infrequent basis The CycLex Res gch Product CycLex PKC Super Family Kinase Assay Kit uses a peroxidase coupled CER aaa phosphoserine monoclonal antibody as a reporter molecule in a 96 well ELISA form is assay provides a non isotopic sensitive and specific method to detect PKC activity The Cyc Research Product CycLex PKC Super Family Kinase Assay Kit is designed to accuratel ermine the prese
26. ubate at 30 Stop the reaction by flicking out the contents Alternatively iffyreaction may be terminated by the addition of 150 uL 0 1 M Na EDTA pH 8 0 to each well Wash wells five times with Wash Buffer making su ch well is filled completely Remove residual Wash Buffer by gentle tapping or aspiration o Pipette 100 uL of Anti Phospho CPI 17 T38 oclonal Antibody into each well cover with plate sealer or lid and incubate at room E ature ca 25 C for 30 minutes Discard any unused antibody after use Wash wells five times as same as in step ow Pipette 100 pL of HRP conjugated fff mouse IgG into each well cover with plate sealer or lid and incubate at room temperature a 25 C for 30 minutes Discard any unused conjugate after use o 10 Wash wells five times as sap in step 6 11 Add 100 uL of Subse am to each well and incubate at room temperature ca 25 C for 5 15 minutes 12 Add 100 uL of SQ olution to each well in the same order as the previously added Substrate Reagent 13 Measure abgerbance in each well using a spectrophotometric plate reader at dual wavelengths of 450 540 n ual wavelengths of 450 550 or 450 595 nm can also be used Read the plate at 450 nm if only single wavelength can be used Wells must be read within 30 minutes of adding the Stop Softition Recoygieindations o rder to estimate the inhibitory effect on PKC activity in the test chemicals correctly it is ne
27. ure the distinct PKC isoenzymes are groupedginto three subfamilies Members of the Ca dependent subfamily are termed conventional or cPKGQynd include PKCa the two alternatively spliced forms of the D gene PKC fI and BIJ and PK 6 These isoenzymes contain three conserved domains namely the diacylglycerol binding ain which contains two repeats of a cysteine rich zinc finger like domain the phospholipid an binding C2 domain and the catalytic C3 C4 domains Members of the second subfamil rmed novel or nPKC are Ca independent for their activity and include PKC 6 e n 0 an 7 10 The C2 like N terminal domain of these isoenzymes can bind acidic phospholipids but no Q A third PKC subfamily termed atypical or aPKC includes only two members PKC and v t ossess a single cysteine rich domain which cannot bind phospholipids or phorbol esters 7 11 12 Regulation of PKC activity is mediated by defined cofactQte that interact with specific regions in its regulatory domain 13 as well as transphosphoryl io by serine threonine kinases 14 and autophosphorylation 15 The activation is accompanie removal of the basic pseudosubstrate region from the kinase domain 16 and may involve the assogfation with specific proteins termed receptors for activated PKC 17 that were suggested to functioaQi selective scaffolds for activated PKC at discrete subcellular compartments b gt Distinct PKC isoenzymes exhibit differen On tissue dis
28. xis MN nnn Assay Characteristics gt The CycLex Research Product CycLex PKC Super Family ase Assay Kit has been shown to detect the PKC activity in column fractions of human or animaktissue lysates The assay shows good linearity of sample response The assay may be used to follow dhe purification of PKC It should be noted that this assay kit detects not only GRC activity but also other protein kinase activities e g Protein kinase A activated by partial proteolysis Rho kinase and ILK in crude extract and column sample in the absence of Ca phospholipid Troubleshooting 1 All samples and controls should be assayedn duplicate using the protocol described in the Detailed Protocol Incubation times or temper significantly different from those specified may give erroneous results e 2 The reaction curve is nearly a strai ff line if the kinetics of the assay is of the first order Variations in the protocol can lead to non lin of the curve as can assay kinetics that are other than first order For a non linear curve point t nt or quadratic curve fit methods should be used 3 Poor duplicates accompaff lj by elevated values for wells containing no sample indicate insufficient washing If all instructiafis in the Detailed Protocol were followed accurately such results indicate a need for washer main ce 4 Overall low sign ay indicate that desiccation of the plate has occurred between the final wash and addition of Substrat

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