Data Sheet
Contents
1. Solvent Inhibitor Assay reagents Test sample control sontrol Kinase Reactiombuffer 80 uL 80 uL 80 uL 10X Inhibitor r equivalent 10 uL Solvent forInhibitor 10 pL 10X Staurosporine 1 M 10 uL PDK 1 positive control 1 m unit nL 10 uL 10 uL 10 uL or Purified enzyme sample Cat S 4400 See Page 4 section Materials Required but not Provided PDKI positive control Cat CY E1180 See Page 4 section Materials Required but not Provided D Following the above table add the Reagents to each well of the microplate Finally initiate reaction by adding 10 uL of PDK1 positive control to each well and mixing thoroughly at room temperature Cover with plate sealer or lid and incubate at 30 C for 30 minutes Cat CY 1180 8 Version 120420 yy wyclex User s Manual 2 Follow the Standard Assay steps 5 12 page 6 7 Special considerations when measuring precise PDK1 activity PDK1 Kinase Assay Inhibitor Screening Kit For Research Use Only Not for use in diagnostic procedures In order to measure the activity of PDK1 correctly it is necessary to conduct the control experimentof 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 Althoughsthe level of A450 increases in Test sample when PDK1 enzyme activity is in the sample the hi2. 50 100 150 ATP conc uM lt s gt Fig 4 Effect of broad spectrum kinase inhibitor staurosporine on PDK1 activity 110 p 100 NU Baan Oo oD oOo o o o o o Rerated intensity of control eee ane E Lb tiiiitl 11 titit a 0 1 1 10 100 1000 Staurosporine nM Cat CY 1180 12 Version 120420 P PDK1 Kinase Assay Inhibitor Screening Kit oy cdl ycLex User s Manual For Research Use Only Not for use in diagnostic procedures References j Toker A and Newton AC 2000 Cellular signaling pivoting around PDK 1 Cell 103 185 188 2 Mora A Komander D van Aalten DMF Alessi DR 2004 PDK1 the master regulator of AGC kinase signal transduction Semin Cell Dev Biol 15 161 170 U Alessi DR James SR Downes CP Holmes AB Gaffney PR Reese CB Cohen PA 1997 Characterization of a 3 phosphoinositide dependent protein kinase which phosphorylates and activates protein kinase Balpha Curr Biol 7 261 269 4 Stephens L Anderson K Stokoe D Erdjument Bromage H Painter GF Holmes AB Gaffney PR Reese CB McCormick F Tempst P Coadwell J Hawkins PT 1998 Protein kinase B kinases that mediate phosphatidylinositol 3 4 5 trisphosphate dependent activation of protein kinase B Science 279 710 714 5 Currie RA Walker KS Gray A Deak M Casamayor A Downes CP Cohen P Alessi DR Lucocq J 1999 Role of phosphatidylinositol 3 4 5 trisphosphate in regulating th
3. afid P labeled ATP The reaction is terminated by spotting a sample onto a phosphocellulose P81 filter paper disc followed by washing extensively to remove unincorporated radiolabel and the_incorporated radioactivity on P81 filter is counted While sensitive this method is 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 PDK1 Kinase Assay Inhibitor Screening Kit uses anti phospho AKT T308 polyclonal antibody PPT 10 and peroxidase coupled anti rabbit IgG 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 PDK Cat CY 1180 2 Version 120420 P PDK1 Kinase Assay Inhibitor Screening Kit oy cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Principle of the Assay The CycLex Research Product CycLex PDK1 Kinase Assay Inhibitor Screening Kit is a single site semi quantitative immunoassay for PDK1 activity Plates are pre coated with a substrate peptide corresponding to surrounding AKT1 T308 which contains threonine residues that can be efficiently phosphorylated by PDK1 The detector antibody specifically detects only the phosphorylated form of threonine residue on substrate peptide The CyeLex PDK1 Kinase Assay Inhibitor Screening Kit may be u
4. PI3 kinase 6 PDK1 seems to exist in an active phosphorylated configuration under basal conditions and appears to be refractive to additional activation and phosphorylation upon cell stimulation with agonists that activate PI3 kinase 7 9 In unstimulated cells overexpressed PDK1 is mainly cytosolic with some localization at the plasma membrane 10 11 PH domain mutants of PDK1 that do not interact with 3 PIs are entirely cytosolic indicating that the membrane association of PDK1 is dependent upon a functional PH domain 11 It has been reported that high level of activated Akt is present in a large percentage 30 60 of common tumor types including melanoma and breast lung gaStric prostate hematological and ovarian cancers When activated in tumor cells Akt has multiple effects that promote disease progression including suppression of apoptosis and stimulation of tumor cell proliferation metabolism and angiogenesis 12 13 The PDK1 Akt signaling pathway plays a key role in cancer cell growth survival and tumor angiogenesis and thus represents an attractive target for the development of small molecule inhibitors that may be useful in the treatment of cancer Measurement of PDK1 activity The protocol generally regarded as most sensitive for the quantitative measurement of PDK1 activity involves incubation of the PDK1 sample with substrate either a natural or synthetic polypeptide such as PDKtide in the presence of Mg
5. as same as in step 6 9 Pipette 100 uL of HRP conjugated Anti rabbit IgG into each well cover with plate sealer or lid and incubate at room temperature ca 25 C for 30 minutes Discard any unused conjugate after use 10 Wash wells five times as same as in step 6 11 Add 100 uL of Substrate Reagent to each well and incubate at roomptemperature ca 25 C for 5 15 minutes 12 Add 100 uL of Stop Solution to each well in the same order as the previously added Substrate Reagent 13 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 activatorsjor inhibitors In order to estimate the inhibitory effect on individual PDK 1 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 forthe first experiment in addition to Test sample as indicated in the following table When test chemicals cause an inhibitory effect on PDK1 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
6. the phosphorylation reaction the absorbance value should be greater than 1 0 with a background less than 0 15 2 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 PDK1 Kinase Assay Inhibitor Screening Kit has been shown to detect the activity of purified recombinant PDK1 The assay shows good linearity of sample response Troubleshooting The CycLex PDK1 positive control Cat CY E1180 should be run in duplicate when a standard assay is being performed using the protocol describedfin the Detailed Protocol Incubation times or temperatures significantly different from those specified may give erroneous results p 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 U 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 was
7. 450 nm Cat CY 1180 3 Version 120420 pry PDK1 Kinase Assay Inhibitor Screening Kit ycLex 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 AKT308 peptide as PDK1 substrate 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 Kinase Reaction Buffer and sample dilution 20X ATP Lyophilized ATP Nay salt Anti Phospho AKT T308 Polyclonal Antibody PPT 10 One vial containing 12 mL of anti phospho AKT T308 polyclonal antibody PPT 10 Ready to use HRP conjugated Anti rabbit IgG One vial containing 12 mB of HRP horseradish peroxidase conjugated anti rabbit IgG Ready to use Substrate Reagent One bottle containing 20 mL offthe chromogenic substrate tetra methylbenzidine TMB Ready to use Stop Solution One bottle containing 20 mL of 1 N H2SOx Ready to use Materials Required but not Provided e PDK1 Positive Control Available from CycLex PDK1 Positive Control Cat CY E1180 One vial containing 4 units 100 uL PDK1 enzyme Positive control should be ad
8. 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 U To assay partially purified recombinant PDK1 add 10 uL of each fraction to the wells of the assay plate on ice Duplicate wells containing 20 m units 10 uL of PDK1 positive control Cat CY E1180 should be included in each assay as a positive control for phosphorylation 4 Begin thekinase reaction by addition of 90 uL Kinase Reaction buffer per well cover with plate sealer and incubate at 30 C for 30 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 an Pipette 100 uL of Anti Phospho AKT T308 Polyclonal Antibody PPT 10 into each well cover with plate sealer or lid and incubate at room temperature ca 25 C for 30 minutes Cat CY 1180 6 Version 120420 p PDK1 Kinase Assay Inhibitor Screening Kit oy cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 7 Wash wells five times as same as in step 5 8 Pipette 100 uL of HRP conjugated Anti rabbit IgG into each well cover with plate sealer or lid an
9. d incubate at room temperature ca 25 C for 30 minutes Discard any unused conjugate after use 9 Wash wells five times as same as in step 5 10 Add 100 uL of Substrate Reagent to each well and incubate at room temperature ca 25 C for 5 15 minutes 11 Add 100 pL of Stop Solution to each well in the same order as the previously added Substrate Reagent 12 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 beqused Read the plate at 450 nm if only a single wavelength can be used Wells must be read within330 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 PDK1 positive control Note 3 If the microplate reader is not capable of reading absorbance greater than the absorbance of the PDK1 positive control perform a second reading at 405 nm A new O D values measured at 405 nm is used to determine PDK activity of off scale samples The readings at 405 nm should not replace the on scale readings at 450 nm Kinetic Assay j Remove the appropriate numberof microtiter wells fro
10. ded to the first well at 20 m units well For instance diluted positive control 1 20 use 10 uL for 1 assay Unused PDK1 enzyme should be stored in aliquots at 70 C e 10X Staurosporine 1 4M Staurosporine is available from Sigma Cat S 4400 1 mM stock solution DMSO diluted 1 1 000 im Kinase Buffer Pipettors 2 20 uL 20 200 nL and 200 1000 uL precision pipettors with disposable tips Precision repeating pipettor e Wash bottle or multichannel dispenser for plate washing e Microcentrifuge and tubes for sample preparation e Vortex mixer e Plate reader capable of measuring absorbance in 96 well plates at dual wavelengths of 450 nm 540 nm Dual wavelengths 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 mI graduated cylinder e Reagentyreservoirs Deionized water of the highest quality Disposable paper towels Cat CY 1180 4 Version 120420 PDK1 Kinase Assay Inhibitor Screening Kit 4 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Precautions and Recommendations e Store the PDK1 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 e Allow all the components to come to room temperature before use e All microplate strips that are not immediately r
11. e activity and localization of 3 phosphoinositide dependent protein kinase 1 Biochem J 337 Part 3 575 583 an Vanhaesebroeck B Leevers SJ Ahmadi K Timms J Katso R Driscoll PC Woscholski R Parker PJ Waterfield MD 2001 Synthesis and function of 3 phosphorylated inositol lipids Annu Rev Biochem 70 535 602 7 Alessi D R Deak M Casamayor A Caudwell F B Morrice N Norman D G Gaffney P Reese C B MacDougall C N Harbison D et al 1997 3 Phosphoinositide dependent protein kinase 1 PDK1 structural and functional homology with the Drosophila DSTPK61 kinase Curr Biol 7 776 789 o0 Pullen N Dennis P B Andjelkovi M Dufner A Kozma S C Hemmings B A and Thomas G 1998 Phosphorylation and activatiomof p70s6k by PDK 1 Science 279 707 710 9 Casamayor A Morrice N and Alessi D R 1999 Phosphorylation of Ser 241 is essential for the activity of 3 phosphoinositide dependent protein kinase 1 identification of five sites of phosphorylation in vivo Biochem J 342 287 292 10 Anderson K E Coadwell J Stephens L R and Hawkins P T 1998 Translocation of PDK 1 to the plasma membraneyis important in allowing PDK 1 to activate protein kinase B Curr Biol 8 684 691 11 Currie R A4 Walker K S Gray A Deak M Casamayor A Downes C P Cohen P Alessi D R and Lucocq J 1999 Role of phosphatidylinositol 3 4 5 trisphosphate in regulating the activ
12. eening Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex PDK1 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 PDK1 positive control Cat CY E1180 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 ddH 0 Mix well Store at 4 C for two weeks or 20 C for long term storage 2 Prepare 20X ATP Solution by adding 1 6 mL of ddH O to the vial of 20X ATP provided lyophilized Mix gently until dissolved The final concentration ofahe 20X ATP Solution should be 1 25 mM Store the solution in small aliquots e g 100 uL at 202C 3 Prepare Kinase Reaction Buffer ATP plus by mixing followingwreagents 96 assays 10 assays 1 assay Kinase Buffer provided 9 5 mL 950 uL 95 uL 20X ATP provided 0 5 mL 50 uL 5 pL Total 10mL 1000 uL 100 pL You will need 80 90 uL of Kinase Reaction Buffer ATP plus per assay well Mix well Discard
13. equired should be returned to the zip lock pouch which must be carefully resealed to avoid moisture absorption e Do not use kit components beyond the indicated kit expiration date e Use only the microtiter wells provided with the kit e Rinse all detergent residue from glassware e Use deionized water of the highest quality e 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 e 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 e Avoid contact with Substrate Solution which contains hydrogen peroxide e Avoid contact with Stop Solution which contains Sulfuric Acid e 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 e CAUTION Sulfuric Acid is a strong acid Wear disposable gloves and eye protection when handling Stop Solution Cat CY 1180 5 Version 120420 pry PDK1 Kinase Assay Inhibitor Scr
14. gh level of A450 is not observed in Inhibitor control ATP minus control and No enzyme control Test Pat ATP minus Positive No enzyme Assay reagents or Sample control control control control Kinase Reaction buffer 90 pL 80nL 90 pL 90 pL Kinase Buffer ATP minus 90 uL 10X Staurosporine 1 uM 10 uL Purified enzyme sample 10uL 10uL 10uL PDK1 positive control 1 m unit uL 10 uL Buffer 10 uL Cat S 4400 See Page 4 section Materials Required but not Provided PDK1 positive control Cat CY E1180 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 Purified enzyme sample or Buffer to each well and mixing thoroughly at room temperature Cover with plate sealer or lid and incubate at 30 C for 30 minutes 2 Follow the Standard Assay steps 5 12 page 6 7 Cat CY 1180 Version 120420 pry PDK1 Kinase Assay Inhibitor Screening Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Evaluation of Results pa Average the absorbance values for the PDK 1 sample duplicates positive control and all experimental sample duplicate values when applicable When PDK1 positive control 20 m units assay is included as an internal control for
15. h 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 CycLex Research Product CycLex PDK1 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 1180 10 Version 120420 wy PDK1 Kinase Assay Inhibitor Screening Kit y gt X User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Dose dependency of recombinant PDK1 enzyme reaction 3 5 f 3 0 2 5 2 0 A450 1 5 1 0 0 5 0 0 0 10 20 30 40 PDK1 Positive control m units Fig 2 Time course of recombinant PDK1 enzyme reaction 3 5 rp 3 0 2 5 o 2 0 N lt lt 1 5 1 0 0 5 0 0 0 10 20 30 40 50 60 Reaction Time min Cat CY 1180 11 Version 120420 oy PDK1 Kinase Assay Inhibitor Screening Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 3 Km for ATP recombinant PDK1 y 51 672x 816 23 R 0 9932 Km for ATP 15 796 uM
16. ity and localization of 3 phosphoinositide dependent protein kinase 1 Biochem J 337 575 583 12 Hanada M Feng J and Hemmings B A 2004 Biochim Biophys Acta 1697 3 16 13 Shiojima I and Walsh K 2002 Circ Res 90 1243 1250 Cat CY 1180 13 Version 120420 pry PDK1 Kinase Assay Inhibitor Screening Kit if y 3X User s Manual For Research Use Only Not for use in diagnostic procedures Related Products PDK1 Positive control Cat CY E1180 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 cyclex co jp CycLex CirctLex 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 1180 14 Version 120420
17. m 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 To assay partially purified recombinant PDK1 add 10 uL of each fraction to the wells of the assay plate once Duplicate wells containing 20 m units 10 uL of PDK1 positive control Cat CY E1180 should be included in each assay as a positive control for phosphorylation 44 Begin kinase reaction by addition of 90 uL Kinase Reaction Buffer in duplicate per well in timed intervalsy suggested interval is 4 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 Cat CY 1180 T Version 120420 pry PDK1 Kinase Assay Inhibitor Screening Kit i ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 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 Anti Phospho AKT T308 Polyclonal Antibody PPT 10 into each well cover with plate sealer or lid and incubate at room temperature ca 25 C for 30 minutes 8 Wash wells five times
18. orylated by PDK1 Applications of this kit include 1 Screening inhibitors or activators of PDK1 2 Detecting the effects of pharmacological agents on PDK1 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 1180 1 Version 120420 P PDK1 Kinase Assay Inhibitor Screening Kit oy cdl ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction PDK1 3 phosphoinositide dependent protein kinase 1 plays an important role in insulin and growth factor signalling cascades 1 2 It is known to phosphorylate and activate at least 24 protein kinases that belong to the AGC cAMP dependent cGMP dependent protein kinase C family of protein kinases These comprise isoforms of Akt PKB also p70 ribosomal S6 kinase serum and glucocorticoid responsive kinase p90 ribosomal S6 kinase and PKC PDK1 activates these efzymes by phosphorylating a Ser Thr residue in their activation loop PDK1 is a 556 amino acid protein comprising an N terminal Ser Thr kinase catalytic domain residues 71 359 and a C terminal pleckstrinshomology PH domain residues 459 550 3 4 The latter binds with high affinity to the second messenger phosphatidylinositol 3 4 5 trisphosphate 4 5 generated through insulin and growth factor induced activation of
19. pry PDK1 Kinase Assay Inhibitor Screening Kit cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Non Radioisotopic Kit for Measuring PDK1 Activity CycLex PDK1 Kinase Assay Inhibitor Screening Kit Cat CY 1180 Intended WS6 lt savvcacsseavesessanssecenaseckeeseuedecesonss 1 POE AG E SE EEE 1 TAU OT gases pcan ds sdccaptanaciapsiaroctalcanieasage 2 Principle of the Assay cccessceeteeteeeees 3 Materials Provided is icsinsesninsazeeatinivhesptarerssnes 4 Materials Required but not Provided 4 Precautions and Recommendations 5 Detailed Protocol cccccccccceesseceeeesseeeeeees 6 9 Evaluation of Results cccccceceesseeeeeees 10 Assay Characteristics sescessnssisessorsvseennniorveass 10 Troubleshooting carrscenncasyieriedintccaieeattedee 10 Reagent Stability i accscccosanssecenancnscesenteasmnsantions 10 Example of Test Results ccecceeeeeegd I 12 Es a Ustt so OO ee Perr errr err eerrn MA 13 Related Product cccccccccccesseceeceeessnamtiey ste 14 Intended Use The CycLex Research Product CyeLex PDK1 Kinase Assay Inhibitor Screening Kit is designed to measure the activities of purified PDK1 for the rapid and sensitive evaluation of inhibitors using recombinant PDK1 The phospho threonine specific polyclonal antibody used in this assay kit has been demonstrated to recognize thesphospho threonine 308 in AKT1 which is efficiently phosph
20. sed to study the kinetics of a purified PDK1 as well as to screening PDK1 inhibitor or activator To perform the test the sample is diluted in Kinase Buffer pipetted into the wells and allowed to phosphorylate the bound substrate following the addition of Mg and ATP The amount of phosphorylated substrate is measured by binding it with a PPT 10 an anti phospho AKT T308 polyclonal antibody followed by binding with horseradish peroxidase conjugatedianti rabbit IgG 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 PDK1 activity in the sample For kinetic analysis the PDK1 containing sample is added to the wells in 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 sample to the wells Vv Incubate for 30min at 30 C Wash the wells Add 100 uL of AntiePhospho AKT T308 Polyclonal Antibody PPT 10 Vv Incubate for 30 min at room temp Wash the wells Add 100 uL of HRP conjugated anti rabbit IgG y Incubate for 30 min at room temp Wash the wells v Add 100 uL of Substrate Reagent v Add 100 pL of Stop Solution v Measure absorbance at
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