Data Sheet
Contents
1. 9 Add 100 uL of Substrate Reagent to each well and incubate at room temperature ca 259C for 10 15 minutes 10 add 100 uL of Stop Solution to each well in the same order as the previously added Substrate Reagent 11 Measure absorbance in each 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 wavelength can be used Wells must be read within 30 minutes of adding the Stop Solution Recommendations Special considerations when screening activators or inhibitors In order to estimate the inhibitory effect on individual AKT activity in the test chemicals correctly it is necessary to conduct the control experiment of Solvent control at least once for every experiment and Inhibitor control at least once for the first experiment in addition to Test sample as indicated in the following table When test chemicals cause an inhibitory effect on AKT activity the level of A450 is weakened as compared with Solvent control The high level of A450 is not observed in Inhibitor control usually A450 lt 0 3 Assay reagents Test sample Solvent Inhibitor control control Kinase Reaction buffer 80 uL 80 uL 80 uL 10X Inhibitor or equivalent 10 uL Solvent for Inhibitor 10 uL 10X Staurosporine 10 uM 10 uL or your enzyme fraction Cat S 4400 See Page 4 section Materials Required but not E2. 1995 9 Cheng J Q Ruggeri B Klein W M Sonoda G Altomare D A Watson D K and Testa J R Proc Nat Acad Sci 93 3636 3641 1996 10 Toshiyuki Obata Michael B Yaffe German G Leparc Elizabeth T Piro Hiroshi Maegawa Atsunori Kashiwagi Ryuichi Kikkawa and Lewis C J Biol Chem 2 75 36108 36115 2000 11 Vanhaesebroeck B Alessi D R Biochem J 346 561 576 2000 Review 12 Franke T F Kaplan D R and Cantley L C Cell 88 435 437 1997 Review 13 Hemmings B A Science 275 628 630 1997 Review Related Products AKTI Positive control Cat CY E118 1 AKT2 Positive control Cat CY E118 2 Anti phospho AKTide 2T monoclonal antibody Clone AT 3E2 Cat CY M1025 PRODUCED BY CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyclex co jp URL http www eyelex 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 1168 13 Version 120420
3. and Recommendations e Store the AKT enzyme at 70 C and the ATP at 20 C when not in use Store all other components at 4 C Do not expose reagents to excessive light Avoid freeze thaw cycles 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 e The buffers and reagents used in this kit contain either sodium Kathon CG as preservatives 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 e Dispose of tetra methylbenzidine TMB containing solutions in compliance with local regulations Avoid contact with Substrate Solution which contains hydrogen peroxide Avoid contact with Stop Solution which contains Sulfuric Acid e n case of contact with the Stop Solution and the Substrate Solution wash skin thoroughly with water and seek medical attention when necessary e Biological samples may be contaminated with infectious ag
4. e g 100 uL at 20 C 3 Prepare Kinase Reaction Buffer ATP plus by mixing following reagents Kinase Buffer provided 9 5 mL 950 uL 95 uL 20X ATP provided 0 5 mL 50 uL 5 uL You will need 80 90 uL of Kinase Reaction Buffer ATP plus per assay well Mix well Discard any unused Kinase Reaction Buffer ATP plus after use Standard Assay 1 Remove the appropriate number of microtiter wells from the foil 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 be assayed in duplicate 3 Add 10 uL of diluted sample to the well of the assay plate on ice Duplicate wells containing 20 m units 10 uL AKTI Cat CY E1168 1 or AKT2 Cat CY E1168 2 should be included in each assay as a positive control for phosphorylation 4 Begin the kinase reaction by addition of 90 uL Kinase Reaction buffer per well cover with plate sealer and incubate at 30 C for 60 minutes 5 Wash wells five times with Wash Buffer making sure each well is filled completely Remove residual Wash Buffer by gentle tapping or aspiration 6 Pipette 100 uL of HRP conjugated Detection Antibody AT 3E2 into each well cover with a plate sealer and incubate at room temperature ca 25 C for 60 minutes Discard any unused Cat CY 1168 6 Version 120420 AKT PKB kinase Assay Inhibitor Screening Kit vc
5. the foil 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 be assayed in duplicate 3 Add 10 uL of diluted sample to the well of the assay plate on ice Duplicate wells containing 20 m units 10 uL AKTI Cat CY E1168 1 or AKT2 Cat CY E1168 2 should be included in each assay as a positivercontrol for phosphorylation 4 Begin kin se reaction by addition of 90 uL Kinase Reaction Buffer in duplicate per well in timed intervals suggested interval is 5 minutes but should be individually determined for each system After the final addition incubate at 30 C for 20 minutes 5 Stop the reaction by flicking out the contents Alternatively the reaction may be terminated by the addition of 150 uL 0 1 M Na EDTA pH 8 0 to each well 6 Wash wells five times with Wash Buffer making sure each well is filled completely Remove residual Wash Buffer by gentle tapping or aspiration 7 Pipette 100 uL of HRP conjugated Detection Antibody AT 3E2 into each well cover with a plate sealer and incubate at room temperature ca 25 C for 60 minutes Discard any unused Cat CY 1168 7 Version 120420 AKT PKB kinase Assay Inhibitor Screening Kit y YCLE gt 4 User s Manual For Research Use Only Not for use in diagnostic procedures conjugate 8 Wash wells as same as in Step 6
6. 0 uL 10 ob AKT positive control 2 5 m unit uL 10 uL Buffer 10 uL Cat S 4400 See Page 4 section Materials Required but not Provided AKT positive control Cat CY E1168 See Page 4 section Materials Required but not Provided 1 Following the above table add the Reagents to each well of the microplate Finally initiate the reaction by adding 10 uL of Your enzyme fraction or Buffer to each well and mixing thoroughly at room temperature Cover with plate sealer or lid nd incubate at 30 C for 60 minutes 2 Follow the Standard Assay steps 5 10 page 6 7 Evaluation of Results bd Average the absorbance values for the AKT sample duplicates positive control and all experimental sample duplicate values when applicable When AKT positive control 25 m units assay 1s included as an internal control for the phosphorylation reaction the absorbance value should be greater than 1 0 with a background less than 0 2 2 For screening of purification chromatography fractions on graph paper plot the mean absorbance values for each of the samples on the Y axis versus the fraction number on the X axis to determine the location of the eluted purified AKT 3 For kinetic analysis on graph paper plot the mean absorbance values for each of the time points on the Y axis versus the time of each reaction minutes on the X axis Assay Characteristics The CycLex Research Product CycLex AKT PKB k
7. AKT PKB kinase Assay Inhibitor Screening Kit clex User s Manual For Research Use Only Not for use in diagnostic procedures oy Non Radioisotopic Kit for Measuring AKT PKB kinase Activity CycLex AKT PKB kinase Assay Inhibitor Screening Kit Cat CY 1168 Intended Use eene l veu earen EE EEEE EEE l Introduction os dacosie sade rat eecsacenteccadennstateaenssesstaetess 2 Principle of the Assay ssssss 3 Materials Provided 4 Materials Required but not Provided 4 Precautions and Recommendations 5 Detailed Protocol oerte rtr ote 6 9 Evaluation of Results 9 Assay Characteristics ccccccccccceseseseeneees 9 Troubleshooting sse 10 Reagent Stability 0 0 ccceceeeeeeeeeeens 10 Example of Test Results H2 FRE Tet ell CCS ossis ea oesetbr nep od eise sug adsl 13 Related Products eese ecce 13 Intended Use The CycLex Research Product AKT PKB Kinase Assay Inhibitor Screening Kit is designed to measure the activities of purified AKTs for the rapid and sensitive evaluation of inhibitors or activators The phospho serine specific monoclonal antibody used in this assay kit has been demonstrated to recognize the phospho serine residue in AK Tide 2T which is efficiently phosphorylated by AKTs Applications of this kit inclu
8. LM AKT positive control Cat CY E1168 See Page 4 section Materials Required but not Provided 1 Following the above table add the Reagents to each well of the microplate Finally initiate reaction by adding 10 uL of diluted CycLex AKT positive control to each well and mixing thoroughly at room temperature Cover with plate sealer or lid and incubate at 30 C for 60 minutes 2 Follow the Standard Assay steps 5 10 page 6 7 Cat CY 1168 8 Version 120420 AKT PKB kinase Assay Inhibitor Screening Kit oy YCLE gt 4 User s Manual For Research Use Only Not for use in diagnostic procedures Special considerations when measuring precise AKT activity In order to measure the activity of AKT correctly it is necessary to conduct the control experiment of Inhibitor control at least once for every experiment and ATP minus control at least once for the first experiment in addition to No enzyme control as indicated in the following table Although the level of A450 increases in Test sample when AKT enzyme activity is in the sample the high level of A450us not observed in Inhibitor control ATP minus control and No enzyme control Assay reagents Test Inhibitor ATP minus Positive No enzyme Sample control control control control Kinase Reaction buffer 90 uL 80 uL 90 uL 90 uL Kinase Buffer ATP minus 90 uL 10X Staurosporine 10 uM 10 uL Your enzyme fraction 10 uL 1
9. T PKB kinase Assay Inhibitor Screening Kit can be used to study the Kinetics of a purified or partially purified AKT as well as to screening AKT inhibitor or activator To perform the test the sample is diluted in Kinase Buffer pipetted into the wells and allowed to phosphorvlate the bound substrate following the addition of Mg and ATP The amount of phosphorylated substrate is measured by binding it with a horseradish peroxidase conjugate of AT 3E2 a anti phospho AK Tide 2T monoclonal antibody which then catalyzes the conversion of the chromogenic substrate tetra methylbenzidine TMB from a colorless solution to a blue solution or yellow after the addition of stopping reagent The color is quantitated by spectrophotometry and reflects the relative amount of AKT activity in the sample For kinetic analysis the AKT containing sample 1s added to the wells in a similar fashion and at varying times the reaction is stopped by the addition of the chelator sodium ethylenediaminetetraacetate EDTA and the amount of phosphorylated substrate determined as before Summary of Procedure Add 100 uL of reaction to the wells Y Incubate for 60 min at 30 C Wash the wells Y Add 100 uL of HRP conjugated anti phospho AK Tide 2T monoclonal antibody Y Incubate for 60 min at room temp Wash the wells Y Add 100 ulsof Substrate Reagent Y Add00 uL of Stop Solution Y Meas re absorbance at 450 nm Cat CY 1168 3 Version 120420 AKT PKB kina
10. c procedures YCLEX Fig 3 Effect of broad spectrum kinase inhibitor staurosporine on AKT activity IC50 6 uM 100 90 re N x E 80 ua a 10 Be PEL 58 50 O oO O OQ lt ue X ie 40 qc om E s 30 t g E 20 10 0 4 6 0 Z 4 6 8 10 Staurosporine u M Staurosporine uM Cat CY 1168 12 Version 120420 AKT PKB kinase Assay Inhibitor Screening Kit oy cle gt 4 User s Manual For Research Use Only Not for use in diagnostic procedures References bd Staal S P Proc Nat Acad Sci 84 5034 5037 1987 2 Andjelkovic M Alessi DR Meier R Fernandez A Lamb NJ Frech M Cron P Cohen P Lucoeq JM Hemmings BA J Biol Chem 272 31515 24 1997 3 Meier R Alessi D R Cron P Andjelkovic M and Hemmings B A J Biol Chem 272 30491 30497 1997 4 Cross D A E Alessi D R Cohen P Andjelkovi M and Hemmings B A Nature 378 785 789 1995 5 Kohn A D Summers S A Birnbaum M J and Roth R A J Biol Chem 271 31372 31378 1996 6 Dudek H Datta S R Franke T F Birnbaum M J Yao R Cooper G M Segal R A Kaplan D R and Greenberg M E Science 275 661 665 1997 7 Cheng J Q Godwin A K Bellacosa A Taguchi T Franke T F Hamilton T C Tsichlis P N and Testa J R Proc Nat Acad Sci 89 9267 9271 1992 8 Bellacosa A et al Int J Cancer 64 280 285
11. d AKT3 show 81 and 83 amino acid identity with AKTI respectively All AKT isoforms show a broad tissue distribution and consist of an N terminal PH domain a kinase domain and a C terminal regulatory tail Two specific sites one in the kinase domain Thr308 in AKT1 and the other in the C terminal regulatory region Ser473 in AKT1 need to be phosphorylated for full activation of these kinases AKT was among the first proteins known to contain a PH domain a few years before the function of this domain came to light The PH domain of AKT specifically binds PI3K lipid products and a firm link between PI3K and AKT signalling has now been established AKT is cytosolic in unstimulated cells and some of it translocates to the plasma membrane upon activation of PI3K where it becomes activated 2 3 Active AKT then appears to detachyfrom the plasma membrane and to translocate through the cytosol to the nucleus The mechanism of this translocation is unclear AKTI was found to mediate insulin and insulin like growth factor IGF 1 induced cellular responses such as the inhibition of glycogen synthase kinase 3 4 the stimulation of glucose uptake 5 and the promotion of cell survival by inhibiting apoptosis 6 Overexpression of AKT1 or AKT2 is associated with some human ovarian pancreatic and breast carcinomas 7 9 Measurement of AKT activity The protocol generally regarded as most sensitive for the quantitative measurement of AKT activity invol
12. de 1 Screening inhibitors or activators of AKT 2 Detecting the effects of pharmacological agents on AKT activity This assay kit is for research use only and not for use in diagnostic or therapeutic procedures Storage Upon receipt Store all components at 4 C e Don t expose reagents to excessive light Cat CY 1168 1 Version 120420 AKT PKB kinase Assay Inhibitor Screening Kit y YCLE gt 4 User s Manual For Research Use Only Not for use in diagnostic procedures Introduction The AKT oncogene was isolated from the directly transforming murine retrovirus AKT8 which was isolated from an AKR mouse thymoma cell line Staal cloned the human cellular homolog AKT 1 He found a 20 fold amplification of the AKTI gene in 1 of 5 gastric adenocarcinomas tested Phosphoinositide 3 kinases PI3Ks generate specific inositol lipids that have been implicated in the regulation of cell growth proliferation survival differentiation and cytoskeletal changes One of the best characterized targets of PI3K lipid products is the protein kinase AKT or protein kinase B RKB In quiescent cells AKT resides in the cytosol in a low activity conformation Upon cellular stimulation AKT is activated through recruitment to cellular membranes by PI3K lipid products and phosphorylation by 3 phosphoinositide dependent kinase 1 PDK1 Mammals have three closely related AKT genes encoding the isoforms AKTA AKT2 and AKT3 AKT2 an
13. ent immediately after wash Reagent Stability All of the reagents included in the CycLex Research Product CycLex AKT PKB kinase Assay Inhibitor Screening Kit have been tested for stability Reagents should not be used beyond the stated expiration date Upon receipt kit reagents should be stored at 4 C except the ATP must be stored at 20 C Coated assay plates should be stored in the original foil bag sealed by the zip lock and containing a desiccant pack For research use only not for use in diagnostic or therapeutic procedures Cat CY 1168 10 Version 120420 H9 AKT PKB kinase Assay Inhibitor Screening Kit 5 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Dose dependency of recombinant AKT enzyme reaction 60 25 A450 A450 0 5 10 15 20 29 0 5 10 15 20 25 Akt1 positive control m units Akt2 positive control m units Fig 2 Time course of recombinant AKT enzyme reaction 2B Aktl positive control T Akt2 positive control 2 0 20 1 5 1 5 c lt lt 1 lt 1 0 1 0 0 5 0 5 0 0 0 0 0 15 30 45 60 375 90 0 15 30 45 60 75 90 Reaction Time min Reaction Time min Cat CY 1168 11 Version 120420 AKT PKB kinase Assay Inhibitor Screening Kit User s Manual For Research Use Only Not for use in diagnosti
14. ents 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 1168 5 Version 120420 AKT PKB kinase Assay Inhibitor Screening Kit clex User s Manual For Research Use Only Not for use in diagnostic procedures y Detailed Protocol The CycLex AKT PKB kinase Assay Inhibitor Screening 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 AKTI positive control Cat CY E1168 1 or AKT2 positive control Cat CY E1168 2 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 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10X Wash Buffer provided to 900 mL of ddH5 O Mix well Store at 4 C for two weeks or 20 C for long term storage 2 Prepare 20X ATP Solution by adding 0 8 mL of ddH O to the vial of 20X ATP provided lyophilized Mix gently until dissolved the Final concentration of the 20X ATP Solution should be 2 5 mM Store the solution in small aliquots
15. inase Assay Inhibitor Screening Kit has been shown to detect the activity of AKT in column fractions of human or animal cell extracts The assay shows good linearity of sample response The assay may be used to follow the purification of AKT Cat CY 1168 9 Version 120420 AKT PKB kinase Assay Inhibitor Screening Kit oy cle gt 4 User s Manual For Research Use Only Not for use in diagnostic procedures Troubleshooting 1 The CycLex AKT positive control Cat CY E1168 1 2 should be run in duplicate when a standard assay is being performed using the protocol described in the Detailed Protocol Incubation times or temperatures significantly different from those specified may give erroneous results 2 The reaction curve is nearly a straight line if the kinetics of the 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 occ rred between the final wash and addition of Substrate Reagent Do not allow the plate to dry out Add Substrate Reag
16. le gt 4 User s Manual For Research Use Only Not for use in diagnostic procedures conjugate 7 Wash wells five times as same as in step5 8 Add 100 uL of Substrate Reagent to each well and incubate at room temperature ca 25 C for 5 15 minutes 9 Add 100 uL of Stop Solution to each well in the same order as the previously added Substrate Reagent 10 Measure absorbance in each 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 wavelength can be used Wells must be read within 30 minutes of adding the Stop Solution 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 for the blank no enzyme control or 2 5 units for the AKT positive control Note 3 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 405 nm is used to determine AKT activity of off scale samples The readings at 405 nm should not replace the on scaleweadings at 450 nm Kinetic Assay 1 Remove the appropriate number of microtiter wells from
17. se Assay Inhibitor Screening Kit y YCLE gt 4 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 microtiter plate kit 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 AK Tide 2T as an AKT substrate 10X Wash Buffer One bottle containing 100 mL of 10X buffer containing 2 6Tween 20 Kinase Buffer One bottle containing 20 mL of 1 X buffer used for Kinase Reaction Buffer and sample dilution 20X ATP Lyophilized ATP Na salt Reconstitute contents of vial with 0 8 mL of ddH O See section Preparation of Working Solution page 6 HRP conjugated Detection Antibody One vial containing 12 mb of HRP horseradish peroxidase conjugated anti phospho AK Tide 2T AT 3E2 monoclonal antibody Ready to use Substrate Reagent One bottle containing 20 mL of the chr mogenie substrate tetra methylbenzidine TMB Ready to use Stop Solution One bottle containing 20 mL of 1 N H2504 Ready to use Materials Required but not Provided e AKT positive control Available from CycLex AKTI positive control Cat CY E1168 1 AKT2 positive control Cat CY E1168 2 One vial containing 5 units 100 uL AKT enzyme Positive control should be added to the firs
18. t well at 25 m units well For instance diluted positive control 1 20 with enzyme dilution buffer use LO uL for 1 assay Unused AKT enzyme should be stored in aliquots at 70 C The AKTI Positiveycontrol should be diluted with an enzyme dilution buffer to avoid inactivating the enzyme activity in low protein concentration condition e Enzyme dilution buffer Mix 9 parts of Kinase buffer and 1 part of 10X BSA 100 ug mL x 0 25 mL which is supplied with AKT positive control e 10X Staurosporine 10 gM Staurosporine is available from Sigma Cat S 4400 1 mM stock solution DMSO diluted 1 100 in Kinase Buffer e Pipettors 2 20 uL 20 200 pL and 200 1000 uL precision pipettors with disposable tips Precision repeating pipettor Wash bottle or multichannel dispenser for plate washing Microcentrifuge and tubes for sample preparation Vortex mixer Plate read r capable of measuring absorbance in 96 well plates at dual wavelengths of 450 nm 540 nm Dual w velengths of 450 550 or 450 595 nm can also be used The plate can also be read at a single wavelength of 450 nm which will give a somewhat higher reading 500 or 1000 mL graduated cylinder Reagent reservoirs Deionized water of the highest quality Disposable paper towels Cat CY 1168 4 Version 120420 AKT PKB kinase Assay Inhibitor Screening Kit y YCLE gt 4 User s Manual For Research Use Only Not for use in diagnostic procedures Precautions
19. ves incubation of the AKT sample with substrate either a natural or synthetic polypeptide such as AKTide 2T in the presence of Mg and P labeled ATP The reaction is terminated by spotting a sample onto a phospho cellulose P81 filter paper disc followed by washing extensively to remove unincorporated radiolabel and the radioactivity counted While sensitive this method 1s labor intensive generates hazardous radioactive waste and depends on a radioisotope of short half life It is particularly unsuitable when kinase assays are only performed on an infrequent basis The CycLex AKT PKB kinase Assay Inhibitor Screening Kit uses peroxidase coupled anti phospho AKTide 2T monoclonal antibody as a reporter molecule in a 96 well ELISA format This assay provides a non isotopic sensitive and specific method to measure the activities of AKT PKB kinase Cat CY 1168 2 Version 120420 AKT PKB kinase Assay Inhibitor Screening Kit y YCLE gt 4 User s Manual For Research Use Only Not for use in diagnostic procedures Principle of the Assay The CycLex Research Product CycLex AKT PKB kinase Assay Inhibitor Screening Kit is a single site semi quantitative immunoassay for AKT activity Plates are pre coated with AKTide 2T 10 which can be efficiently phosphorylated by AKTI 2 and 3 on a microtiter plate The detector antibody is AT 3E2 an antibody that specifically detects only the phosphorylated AKTide 2T The CycLex AK
Download Pdf Manuals
Related Search