CY-1185
Contents
1. 5 Stop the kinase reaction by addition of 10 uL of EDTA Solution to eachywell 6 Transfer 100 uL of the reaction mixture in each well of the Non eoated Microplate to each well of the Antibody coated Microplate cover with plate sealer and incubate at room temperature ca 25 C for 60 minutes shaking at ca 300 rpm on an orbitalmicroplate shaker 7 Wash wells five times with Wash Buffer making s re each well is filled completely Remove residual Wash Buffer by gentle tapping or aspiration 8 Pipette 100 uL of HRP conjugated Detection Antibody to each well cover with the lid and incubate at room temperature ca 25 C for 60sminutes shaking at ca 300 rpm on an orbital microplate shaker o Wash wells five times as same as in step 7 1 gt Add 100 uL of Substrate Reagent to cach well and incubate at room temperature ca 25 C for 5 20 minutes shaking at ca 300 rpmoon an orbital microplate shaker 11 Add 100 uL of Stop Solution to each well in the same order as the previously added Substrate Reagent 12 Measure absorbance imeach well using a spectrophotometric plate reader at dual wavelengths of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used Read the plate at 450 nm if only a single wayelength can be used Wells must be read within 30 minutes of adding the Stop Solution Cat CY 1185 9 Versions 141107 oe CaMKK beta Kinase Assay Kit e Vclex User s Manual For Research Use Only No2. by adding 10 ul of CaMKKf sample or Positive Control or Buffer for CaMKKp sample to each well and mixing thoroughly at room temperature Cover with plate lid and incubate at 30 C for 60 minutes shaking at ca 300 rpm on an orbital microplate shaker 2 Follow the steps 5 12 of Standard Assay on page 9 Note Although we suggest to conduct experiments as outlined in the table above the optimal experimental conditions will vary depending on the parameters being investigated and must be determined by the individual user Especially an appropriate amount of the enzyme must be optimized by titration of the enzyme and setting the amount which shows OD value does not exceed plateau range in dose response curve Cat CY 1185 11 Version 141107 H9 CaMKK beta Kinase Assay Kit e Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Evaluation of Results Average the absorbance values for the Positive Control and all experimental sample duplicate values when applicable When Positive Control See 7 Prepare Positive Control for kinase reaction on page 7 is included in 50 units assay as an internal control for the kinase reaction the absorbance value should be greater than 1 0 with a background less than 0 25 when using Reaction buffer Assay Characteristics The CycLex Research Product CycLex CaMKK beta Kinase Assay Kit has been sli wn to detect the activity of purified CaMKKfp a
3. solution to a blue solution or yellow after the addition of stopping reagent The color is quantified by spectrophotometry and reflects the relative amount of CaMKKp activity in the sample For kinetic analysis the CaMKKf containing sample is added to the wells in a similar fashion and at varying times the reaction is stopped by the addition ofthe chelator sodium ethylenediaminetetraacetate EDTA and the amount of phosphorylated substrate determined as before The CycLex Research Product Cycbex CaMKK beta Kinase Assay Kit is designed to accurately determine the presence and relative amount of CaMKKf activity in purification column fractions and to determine non isotopic kinetic analysis of CaMKK activity Careful attention to extraction methods and the assay protocol will providefithe investigator with a reliable tool for the evaluation of CaMKKf activity Cat CY 1185 2 Version 141107 Fg CaMKK beta Kinase Assay Kit yclex User s Manual For Research Use Only Not for use in diagnostic procedures Summary of Procedure 2 Add 100 uL of reaction mixtures to the non coated wells uus Incubate for 1 hour at 30 C Men cubate for 1 hour at AMPKal T183 Add of 10 uL of EDTA Solution to stop kinase O Transfer 100 uL of reaction mixtures to the antib e wells Incubate for 1 hour at room temp anti phospho AMPKal T183 AMPKo2 T172 monoclonal antibody Wash the wells Add 100 uL of HRP conjugated anti AM
4. 11 Mix the following reagents 96 assays 10 assays 1 assay Kinase Buffer provided 7 2 mL 720 uL 72 uL 10X AMPKal Substrate Solution 1 0 mL 100 uL 10 uL 20X DTT Solution 0 5 mL 50 uL 5uL 20X BSA Solution 0 5 mL 50 uL 5uL 50X EGTA provided 0 2 mL 20 uL 2uL ddH O 0 6 mL 60 uL 6 uL Total 10 mL 1 000 uL 100 pL 60 90 uL of Reaction Buffers per assay well will be needed Mix well Discard any unus d Reaction Buffers after use Cat CY 1185 8 Version 141107 oe CaMKK beta Kinase Assay Kit e Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Standard Assay 1 Remove the appropriate number of wells of Non coated Microplate and Antibody coated Microplate from the pouch and place them into the well holder Return any unused wells to the foil pouch refold seal with tape and store at 4 C 2 Prepare all samples diluted with Kinase Buffer as needed All samples should b assayed in duplicate 3 Add 10 uL of sample and Positive Control See 7 Prepare Positive Control for phosphorylation on page 7 to each well of Non coated Microplate on ice The Positive Control should be included in duplicate wells in each assay 4 Start the kinase reaction by addition of 90 uL Reaction buffer Ca CaM_ plus to each well of the Non coated Microplate cover with plate sealer and incubate at 30 C for 60 minutes shaking at ca 300 rpm on an orbital microplate shaker
5. 41107 CaMKK beta Kinase Assay Kit a yclex User s Manual For Research Use Only Not for use in diagnostic procedures 2 Add protein A agarose beads 20 uL of 50 bead slurry Incubate with gentle rocking for hours at 4 C 3 Microcentrifuge for 30 seconds at 4 C Wash the protein A agarose beads 3 times with 500 uL of Cell Lysis Buffer and successively once with Kinase Buffer Keep on ice during washes X 4 Resuspend the protein A agarose beads with 20 40 uL of Kinase Buffer and use 10 u an enzyme sample to measure CaMKKp activity according to the procedure in the step 3 12 of Standard Assay on page 9 Please take care to transfer the protein A agarose beads to the well of t tibody coated Microplate as little as possible C CY 1185 14 Versionft 141107 Fg CaMKK beta Kinase Assay Kit es yclex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Dose dependency of recombinant CAMKKp 3 0 A ATP 2 5 2 0 215 lt lt 1 0 0 5 0 0 2 rd tt A p tt pic qp p pepe 0 20 40 60 80 100 GST CaMKKf units Fig 2 Km for ATP of recombinant CAaMKKp 12 000 y 55 665x 241 53 10 000 R 0 9991 8 000 Km 4 3 uM 6 000 4 000 2 000 OD450 min s v 0 fi L fi 1 1 1 1 0 25 50 75 100 125 150 175 200 ATP conc uM s C CY 1185 15 Ver
6. Edelman AM Frenguelli BG Hardie DG Gell Metab 2005 2 1 9 19 2 Shaw RJ Kosmatka M Bardeesy N Hurley RL Witters LA DePinho RA Cantley LOL PNAS USA 2004 101 10 3329 3335 3 Hawley S Boudeau J Reid J Mustard K Udd L Makela T Alessi D Hardie DG J Biol 20035 2 4 28 4 Hurley RL Anderson KA Franzone JM Kemp BE Means AR Witters LA JBG 2005 280 32 29060 29066 5 Hawley SA Pan DA Mustard KJ Ross L Bain J Edelman AM Frenguellt BG Hardie DG Cell Metab 2005 2 1 9 19 6 Woods A Dickerson K Heath R Hong S P Momcilovic M Johnstone SR Carlson M Carling D Cell Metab 2005 2 1 21 33 7 Anderson KA Means RL Huang Q H Kemp BE Goldstein EG Selbert MA Edelman AM Fremeau RT Means AR JBC 1998 273 48 31880 31889 Related Products CycLex CaMKK beta Kinase Assay Kit Cat CY 1185 CaMKK beta Positive Control Full length z at CY E1185 2 PRODUCED BY CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 481 265 7647618 e mail info cyclex co jp URL http www cyclex co jp CycLex CircuLex products are supplied for research use only CycLex CircuLex products and components thereof may not be resold modified for resale or used to manufacture commercial products without prior written approval from CycLex Co Ltd To inquire about licensing for such commercial use please contact us via email Cat CY 1185 18 Versions 141107
7. L 10X Inhibitor or equivalent 10 pL Vehicle for inhibitor 10 uL 10X STO 609 20 uM 10 pL Positive Control 10 uL 10 uL 10 uL or CaMKKf sample See Materials Required but n t Provided on page 5 5 units uL of CaMKK beta ositive Control Full length See Materials Required but not Provided on page 5 and 7 Prepare Positive Control for kinase reaction on page 7 n Following the above table add the Reagents to each well of the microplate Finally initiate reaction by adding 10 uL of Positive Control or CaMKKf sample to each well and mixing thoroughly at room temperature Cover with plate sealer or lid and incubate at 30 C for 60 minutes shaking at ca 300 rpm onamorbital microplate shaker 2 Follow the steps 5 12 of Standard Assay on page 9 Note Although we suggest to conduct experiments as outlined in the table above the optimal Cat CY 1185 10 Versions 141107 v oy CaMKK beta Kinase Assay Kit Vclex User s Manual For Research Use Only Not for use in diagnostic procedures experimental conditions will vary depending on the parameters being investigated and must be determined by the individual user Especially an appropriate amount of the enzyme must be optimized by titration of the enzyme and setting the amount which shows OD value does not exceed plateau range in dose response curve Special considerations for measuring precise CAMKK f a
8. PKa antibody Q hour at room temp Wash e e of Substrate Reagent dd 100 uL of Stop Solution G absorbance at 450 nm C CY 1185 3 Versions 141107 oe CaMKK beta Kinase Assay Kit e Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Materials Provided All samples and standards should be assayed in duplicate The following components are supplied and are sufficient for the one 96 well microplate kit Non coated Microplate One microplate supplied ready to use for kinase reaction with 96 wells 12 strips of 8 wells in a clear zip lock bag Antibody coated Microplate One microplate supplied ready to use with 96 wells 12 strips of 8 wells in a foil zip lock bag with a desiccant pack Wells are coated with anti phospho AMPKal T183 AMPKo2 T172 monoclonal antibody as a capture antibody 10X Wash Buffer One bottle containing 100 mL of 10X buffer containing 2 Tween 20 Kinase Buffer One bottle containing 20 mL of 1X buffer used for Reaction Buffer and sample dilution 20X ATP One vial of lyophilized ATP Naz salt 20X DTT Two vials of lyophilized dithiothreitol 20X BSA One vial of lyophilized BSA 100X Calmodulin One vial of lyophilized calmodulin us d for Reaction Buffer Ca CaM plus 50X CaCb One vial containing 0 4 mL of 125 mM CaCl used for Reaction Buffer Ca CaM plus 50X EGTA One vial containing 0 4 mL of 100 mM EGTA used for Reaction Buffer Ca CaM min
9. ctivity In order to measure the activity of CaMKKf correctly it is necessary to conduct the control experiment of Inhibitor control at least once for every experiment and Ca CaM minus control and ATP minus control at least once for the first experiment in addition to No enzyme Contfol as indicated in the following table Although the level of A450 increases in Test sample when CaMKKf enzyme activity is in the sample the high level of A450 is not observed in Inhibit6fcontrol ATP minus control and No enzyme control 2 Test Inhibitor JCaM AEE Positive No Assay reagents minus minus enzyme sample control control control control control Reaction buffer Ca CaM plus 80 pL 80 uL 80 uL 80 uL Reaction buffer Ca CaM minus 80 uL Reaction buffer Ca CaM ATP minus 80 uL 10X STO 609 20 uM 10 pL Vehicle for 10X STO 609 10 pL 10 uL 10uL 10 uL 10 pL CaMKKf sample 10 pL 10 uL 10 pL 10 pL Positive Control 10 uL Buffer for CAaMKKfp sample 10 uL See Materials Required but not Provided on page 5 units uL of CaMKK beta Positive Control Fullylength See Materials Required but not Provided on page 5 and 7 Prepare Positive Control for kinase reaction on page 7 1 Following the above table add th Reagents to each well of Non coated Microplate Finally initiate the reaction
10. d be traced by other methods e g western blotting Immunoprecipitation Protocol Followed by Measuring CaMKKf Activity Preparation of Solution and Reagent A Preparation of Cell Lysis Buffer 20 mM Tris HCl pH 7 5 250 mM NaCl 10 96 glycerol 0 5 Nonidet P 40 1 mM EDTA 1 mM EGTA 0 2 mM PMSF 1 ug mL pepstatin 0 5 ug mL leupeptin 5 mM NaF 2 mM NasVO4 2 mM B glycerophosphate 1 mM DTT B Preparation of Protein A Agarose Beads Add 5 mL of 1X PBS to 1 5 g of protein A agarose beads Shake 2 hours at 4 C spin down Wash beads twice with PBS Resuspend beads in 1 volume of PBS Can be stored for 2 weeks at 4 C Preparing Cell Lysates pa Plate adherent cells in 10cm dish plat amp t JO x 10 cells plate and incubate the plate at 37 C for 12 16 hours in CO incubator 2 Remove media and wash cells with ice cold PBS and aspirate Uo Add 1 mL of ice cold Cell Eysis Buffer to the plate and incubate on ice for 5 minutes 4 Scrape off and transfer fhe lysate to a microcentrifuge tube Nn Rotate the tube for 60 minutes at 4 C and microcentrifuge at 15 000 rpm for 10 minutes at 4 C 6 Transfer the Supernatant to a new tube The supernatant is the cell lysate If necessary the lysate can be stored at 0SC Immunoprecipitation 1 Take 100 uE cell lysate and add anti CaMKKp antibody Abcam Cat ab168818 1 2 ug incubate with gentleyrocking for 2 hours or overnight at 4 C Cat CY 1185 13 Version 1
11. e the solution in small aliquots e g 100 uL at 20 C 7 Prepare Positive Control for kinase reaction Dilute the CaMKK beta Positive Control Full length CycLex Co Ltd Cat CY E1185 2 see page 5 to the final con entration of 5 units uL using Kinase Buffer provided Cat CY 1185 7 Version 141107 Pg YCLEX 8 Prepare Reaction Buffer for kinase reaction Reaction Buffer Ca CaM plus Mix the following reagents CaMKK beta Kinase Assay Kit User s Manual For Research Use Only Not for use in diagnostic procedures 96 assays 10 assays 1 assay Kinase Buffer provided 7 2 mL 720 uL 72 pL 10X AMPKa1 Substrate Solution 1 0 mL 100 uL 10 uE 20X ATP Solution 0 5 mL 50 uL 5 uL 20X DTT Solution 0 5 mL 50 uL 5 uL 20X BSA Solution 0 5 mL 50 uL SuL 50X CaCh provided 0 2 mL 20 uL 2 uL 100X Calmodulin provided 0 1 mL 10 pL luL Total 10 mL 1 000 4 100 pL Reaction Buffer Ca CaM minus for measuring precise CaMKK activity See page 11 Mix the following reagents 96 assays 10 assays 1 assay Kinase Buffer provided 7 2 mk 720 uL 72 uL 10X AMPKal Substrate Solution 1 0 mE 100 uL 10 uL 20X ATP Solution 0 5 mL 50 pL 5 uL 20X DTT Solution 0 5 mL 50 pL 5 uL 20X BSA Solution 0 5 mL 50 uL 5uL 50X EGTA provided 0 2 mL 20 uL 2 uL ddH20 0 1 mL 10 uL luL Total 10 mL 1 000 uL 100 uL Reaction Buffer Ca CaM ATP mins fo measuring precise CaMKKp activity See page
12. ffer provided to 900 mL of deionized distilled water ddH2O Mix well Store at 4 C for two weeks or 20 C for long term storage N Prepare 20X ATP Solution by adding 1 6 mL of ddH20 to the vial of 20X ATP provided lyophilized Mix gently until dissolved The final concentration of the 20X ATP Solution should be 1 25 mM Store the solution in small aliquots e g 200 uL at 20 C Uo Prepare 20X DTT Solution by adding 0 5 mL of ddH O to the vial of 20X DTT provided lyophilized Mix gently until dissolved The final concentratiom of the20X DTT Solution should be 100 mM Store the solution in small aliquots e g 100 uL at lt 20 C 4 Prepare 20X BSA Solution by adding 0 75 mL of ddHsO to the vial of 20X BSA provided lyophilized Mix gently until dissolved The final concentration of the 20X BSA Solution should be 0 067 mg mL Store the solution in small aliquots e g 100 QD at 20 C Nn Prepare 100X Calmodulin Solution by adding 0 125 mL of ddH20 to the vial of 100X Calmodulin provided lyophilized Mix gently until dissolved The final concentration of the 100X Calmodulin Solution should be 25 ug mL Store the solution in small aliquots e g 50 uL at 80 C lon Prepare 10X AMPKa1 Substrate Solution by adding 1 2 mL of ddH20 to the vial of 10X AMPKa1 Substrate provided lyophilized Mix gently until dissolved The final concentration of the 10X AMPKa1 Substrate Solution should be 31 25 ug mL Stor
13. m Dual wavelengths of 450 550 or 450 595 nm can lso ed The plate can also be read at a single wavelength of 450 nm which will give a s a r reading Software package facilitating data generation a alysis optional 500 or 1000 mL graduated cylinder Reagent reservoirs Deionized water of the highest R O e Disposable paper towels e C CY 1185 5 Versions 141107 oe CaMKK beta Kinase Assay Kit e ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Precautions and Recommendations Allow all the components to come to room temperature before use All microplate strips that are not immediately required should be returned to the zip lock pouch which must be carefully resealed to avoid moisture absorption Do not use kit components beyond the indicated kit expiration date Use only the microtiter wells provided with the kit Rinse all detergent residue from glassware Use deionized water of the highest quality Do not mix reagents from different kits The buffers and reagents in this kit may contain preservatives or other chemi als Care should be taken to avoid direct contact with these reagents Do not mouth pipette or ingest any of the reagents Do not smoke eat or drink when performing the assay or in areas where samples or reagents are handled Dispose of tetra methylbenzidine TMB containifig solutions in compliance with local regulations Avoid contact
14. nd column fractions containing CaMKKf The assay mdy be used to follow the purification of CaMKK or may be used to detect the presence of CaMKK in cell lysates Troubleshooting pa The Positive Control should be run in duplicate when a standard assay s being performed using the protocol described in the Detailed Protocol Incubation tim Spor temperatures significantly different from those specified may give erroneous results 2 The reaction curve is nearly a straight line if the kinetics of th assay is of the first order Variations in the protocol can lead to non linearity of the curve as can assay Kinetics Of other than first order For a non linear curve point to point or quadratic curve fit methods should be used 3 Poor duplicates accompanied by elevated values for wells containing no sample indicate insufficient washing If all instructions in the Detailed Protocol were followed accurately such results indicate a need for washer maintenance 4 Overall low signal may indicate that desiccation of the plate has occurred between the final wash and addition of Substrate Reagent Do not allow the plate to dry out Add Substrate Reagent immediately after wash Reagent Stability All of the reagents included in the Cyebex Research Product CycLex CaMKK beta Kinase Assay Kit have been tested for stability Reagents should not be used beyond the stated expiration date Upon receipt kit reagents should be stored a
15. oe CaMKK beta Kinase Assay Kit e Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Non Radioisotopic Kit for Measuring CAMKKp Activity CycLex CaMKK beta Kinase Assay Kit Cat CY 1185 Intended Use rere 1 SURO MP 1 IntroductiOn ccccccesecccceessesseeceseeseesteeees 2 Principle of the ASSAY wes eene ases Vh indes 2 3 Materials Provided sss 4 Materials Required but not Provided 5 Precautions and Recommendations 6 Detailed Protocol ssssse 7 11 Evaluation of Results sss 12 Assay Characteristics 12 Troubleshooting 12 Reagent Stability saceasisssiseascusssacedansivseasaaeueey 12 Sample Preparallom s nsesten e e te e ket 13 Example or Test Result 14 16 References usc eite ier RENS 17 Related Products ssssss I Intended Use The CycLex Research Product CycLex CaMKK beta Kinase Assay Kit is primarily designed to measure the activities of purified Ca calmodulin dependent protein kinase kinase beta CaMKK or recombinant CaMKKf for the rapid and sensitive evaluation of activators or inhibitors The phospho threonine specific monoclonal gantibody used in this assay kit has been demonstrated to recognize the phospho threonine 183 in adeftosine monophosphate activated protein kinase AMPK al the phospho threonine 172 in AMPKo2 which is efficiently pho
16. sions 141107 Fg CaMKK beta Kinase Assay Kit es vclex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 3 Effect of NaCl on the kinase activity of recombinant CaMKKp 2 5 k ATP amp ATP e 2 0 A450 0 100 200 300 400 500 NaCl mM Fig 4 Effect of CAMKKp inhibitor STO 609 on the 9M CaMKKf 100 IC50 700 ng mL 2 uM 90 80 70 60 50 40 30 relative activity control 20 10 0 1 10 100 1 000 10 000 STO 609 ng mL C CY 1185 16 Version 141107 Pat CaMKK beta Kinase Assay Kit NCl ex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 5 Effect of EGTA and Ca Calmodulin on native CaMKKf immunoprecipitated from C2C12 cell 3 5 3 0 2 5 2 0 l 1 0 0 5 09 E m m m BH m m BH m Hi m Ed m ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP To xo f o koc xo ko t4 o EGTA Ca2 CaM EGTA Ca2 CaM EGTA Ca2 CaM EGTA Ca2 CaM Antibody None Antibody Anti CaMKK2 polyAb Abcam ab1 68818 C2C12 cell extract A450 Un LP No Enzyme GST CaMKKB C CY 1185 17 Versions 141107 H9 CaMKK beta Kinase Assay Kit 5 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures References 1 Hawley SA Pan DA Mustard KJ Ross L Bain J
17. sphorylated by CaMKKp Additionally column fractions of cultured primary cells cell lines or tissues can be assayed for CaMKKp activity with the CycLex Research Product CycLex CaMKK beta Kinase Assay Kit if the appropriate dose of CAMKKp sp eific inhibitor e g STO 609 is used Applications of this kit include 1 Screening activators orinhibitors of CaMKKp 2 Evaluating the effects of pharmacological agents on CaMKK activity in vitro 3 Monitoring the p rification of CaMKKp activity This assay kit is for research use only and not for use in diagnostic or therapeutic procedures Storage Upomiiteceipt store all components at 4 C e Don t expose reagents to excessive light Cat CY 1185 1 Versionft 141107 oe CaMKK beta Kinase Assay Kit e Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction The AMP activated protein kinase AMPK is a critical regulator of energy homeostasis and as a potential target for treatment of metabolic diseases as well as cancer 1 Activation of AMPK requires phosphorylation of threonine 172 within the T loop region of the catalytic a2 subunit by the tumor suppressor LKB1 2 3 or the Ca calmodulin dependent protein kinase kinase beta CaMKKf 4 6 CaMKKp can form a complex with and activate AMPK but CaMKKa cannot In addition it was shown that CaMKKB and AMPK associate through their kinase domains and CAMKKf must be in an active conforma
18. t 4 C Antibody coated Microplate should be stored in the original foil bag sealed by the zip loek and containing a desiccant pack For research use only not for use in diagnostic or therapeutic procedures Cat CY 1185 12 Version 141107 oe CaMKK beta Kinase Assay Kit e ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Sample Preparation Numerous extraction and purification methods can be used to isolate CaMKKB The following protocols have been shown to work with a number of different tissues and enzyme sources and are provided as examples of suitable methods Crude samples can frequently be used without dilution while more concentrated or highly purified CaMKKp should be diluted It is strongly advised that the user always perform an initial experiment to determine the proper dilution to be used in subsequent experiments This need not be any more than a single time point assay using serial dilutions of the crude extract cell lysate or sample fraction taken prior to a purification step One eight well strip of the 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 CAUTION It should be noted that this assay kit detects not only CaMKK activity but also other protein kinases in crude extract and column sample The presence of CGaMKKf protein in the samples shoul
19. t for use in diagnostic procedures 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 and blot it against clean paper towels Note 2 Reliable signals are obtained when either O D values do not exceed 0 25 units r the blank no enzyme control or 2 5 units for Positive Control Note 3 If the microplate reader is not capable of reading absorbance greater than the absorbance of Positive Control perform a second reading at 405 nm A new O D values measured at 405 nm is used to determine Positive Control of off scale samples The readingsfap405 nm should not replace the on scale readings at 450 nm Recommendations Special considerations for screening inhibitors In order to estimate the inhibitory effect on individual CaMKK activity in the test chemicals correctly it is necessary to conduct the control experiment of Vehicle control at least once for every experiment and Inhibitor control at least once for the first experim nt im addition to Test sample as indicated in the following table When test chemicals cause an inhibitory effect on CAMKKp activity the level of A450 is weaken as compared with Vehicle control For inhibitor screening Assay reasents Test Sample Vehicle Inhibitor y reag for nhibitor control control Reaction buffer Ca CaM plus 80 uE 80 uL 80 u
20. tion in order to bind AMPK but not to associate with an alternative substrate Ca calmodulin dependent protein kinase IV In contrast to LKB1 the activation of AMPK by CaMKKp does not require an alteration of the ATP AMP ratio but rather occurg in response to an increase in intracellular Ca The expression pattern of CaMKKf in cells and tissueg if more limited than that of LKB1 and is highest in multiple regions of the brain 7 Principle of the Assay The CycLex Research Product CycLex CaMKK beta Kinase AsSsay Kit is a semi quantitative immunoassay for CaMKKp activity This product can be used to d termine the presence of CaMKKf activity in purification column fractions or to follow the kineties of a purified or partially purified CaMKKf protein as well as screening CaMKK inhibitor or activator The protocol for the quantitative measurement of CaAMKKf kinase activity involves incubation of the CaMKKf sample with its substrate AMPKal GST fusion protein in the presence of Mg and ATP followed by transfer this kinase reaction mixture tothe well of microtiter plate which has been pre coated with a monoclonal antibody specific for phospho AMPKal T183 AMPKo2 T172 for trapping only phosphorylated substrate The amount of phosphorylated substrate on the well is measured by a horseradish peroxidase conjugated antibody specific for AMPKal which then catalyzes the conversion of the chromogenic substrate tetra methylbenzidine TMB from a colorless
21. us 10X AMPKa1 Substrate One vial containing 37 5 ng of lyophilized recombinant GST AMPKal 1 394 EDTA Solution One vial containing 2 mDjof0 5 M EDTA pH 8 0 Ready to use HRP conjugated Detection Antibody One bottle containing 12 mL of HRP horseradish peroxidase conjugated anti AMPKa antibody Ready to use Substrate Reagent One bottle containing 20 mL of the chromogenic substrate tetra methylbenzidine TMB Ready to use Stop Solution One bottle containing 20 mL of 1 N H2S014 Ready to use Cat CY 1185 4 Version 141107 iy CaMKK beta Kinase Assay Kit e Vclex User s Manual For Research Use Only Not for use in diagnostic procedures AN Materials Required but not Provided CaMKK beta Positive Control Full length Available from CycLex Co Ltd Cat CY E11 Unused CaMKK beta Positive Control Full length should be stored in aliquots at below 70 C 10X STO 609 20 uM Available from Calbiochem Cat 570250 1 mM stock sol diluted 1 50 in Kinase Buffer Orbital microplate shaker e Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with Qj Precision repeating pipettor Wash bottle or multichannel dispenser for plate washing G5 Microcentrifuge and tubes for sample preparation Q Vortex mixer e Microplate washer optional Manual washing is possible but not preferable Plate reader capable of measuring absorbance in 96 well es at dual wavelengths of 450 nm 540 n
22. with the acidic Stop Solution and Substrate Solution which contains hydrogen peroxide Wear gloves and eye protection when handling mmunodiagnostic 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 medical attention when necessary Biological samples may be contaminated with infectious agents Do not ingest expose to open wounds or breathe aerosols Wear protective gloves and dispose of biological samples properly CAUTION Sulfuric Acid is a Strong acid Wear disposable gloves and eye protection when handling Stop Solution Cat CY 1185 6 Version 141107 v my CaMKK beta Kinase Assay Kit Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex CaMKK beta Kinase Assay Kit is provided with removable strips of wells so the assay can be carried out on separate occasions using only the number of strips required for the particular determination Since conditions may vary running an aliquot of the appropriate positive control see page 5 separately available from CycLex should be included in each assay Disposable pipette tips and reagent troughs should be used for all transfers to avoid cross contamination of reagents or Samples Preparation of Working Solution um Prepare a working solution of Wash Buffer by adding 100 mL of the 10X Wash Bu
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