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CY-1178

1. 3 5 p 3 0 0 10 20 30 40 50 60 Reaction Time min C CY 1178 11 Version 140318 yelex Fig 3 Dose dependency of ATP User s Manual IKK a and B Assay Inhibitor Screening Kit For Research Use Only Not for use in diagnostic procedures 1 4 OD450 0 10 20 30 40 50 60 70 ATP uM 80 90 100 Fig 4 Km for ATP AN 4500 p 3500 p 3000 T 2500 F 2000 F 1500 f 1000 f 500 F OD450 min lt s v gt 4000 y 19 32x 141 24 R 0 9975 Km for ATP 7 3 uM 0 0 50 100 ATP conc uM lt s gt 150 200 12 Version IKK a and B Assay Inhibitor Screening Kit User s Manual wyclex For Research Use Only Not for use in diagnostic procedures Fig 5 Effect of broad spectrum kinase inhibitor K252a on activity of recombinant IKKB 100 90 80 70 60 50 40 30 20 F 10 F 0 raaa Po aan 0 01 0 1 1 10 K252a uM Relative intensity of control C CY 1178 13 Version 140318 IKK a and B Assay Inhibitor Screening Kit F 4 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures References 1 Ghosh S and Karin M 2002 Cell 109 S81 S96 2 Schmitz L M Bacher S and Kracht M 2001 Trends Biochem Sci 26 186 190 3 Naumann M and Scheidereit C 1994 EMBO J 13 4597 4607 4 Brown K Gerstberger S Carlson L
2. The detector antibody specifically detects only the phosphorylated form of IkBa The CyeLex Research Product CycLex IKK a and Assay Inhibitor Screening Kit can be used to study the kinetics of a purified or partially purified IKK as well as to screening these kinases inhibitor To perform the test the sample is diluted in Kinase Buffer pipetted into the wells and allowed to phosphorylate the bound substrate in the presence of Mg and ATP The amount of phosphorylated substrate is measured by binding it with a horseradish peroxidase conjugate of AS 2E8 an anti phospho IKB S32 specific antibody which then catalyzes the conversion of the chromogenic substrategtetra methylbenzidine TMB from a colorless 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 IKK activity in the sample For kinetic analysis the sample containing IKK is added to the wells in asimilar fashion and at varying times the reaction is stopped by the addition of a chelator sodium ethylenediaminetetraacetate EDTA and the amount of phosphorylated substrate determined as before The CycLex Research Product CycLex IKK a and B Assay Inhibitor Screening Kit is designed to accurately determine the presence and relative amount of IKKo and activities to determine non isotopic kinetic analysis of IKKa and f activities Summary of Procedure Add 100 uL of sample to the wel
3. Franzoso G and Siabenlist U 1995 Setence 267 1485 1488 5 Chen Z Haglen J Palombello V J Melandri F Scherer D Ballard D and Maniatis T 1995 Genes Dev 9 1586 1597 6 DiDonato J Mercurio F Rosette C Wi Li J Suyang H Ghosh S and Karin M 1996 Mol Cell Biol 16 1295 1304 7 Alkalay I Yaron A Hatzubai A Jung S Avraham A Gerlitz O Pashut Ievon I and Ben Neriah Y 1995 Mol Cell Biol 15 1294 1301 8 Karin M and Ben Neriah Y 2000 Annu Rev Immunol 18 621 663 9 DiDonato J A Mercurio F and Karin M 1995 Mol Cell Biol 15 1302 43 4 10 Scherer D C Brockman J Chen Z Maniatis T and Ballard D 1995 Proc Natl Acad Sci U S A 92 11259 11263 11 Spencer E Jiang J and Chen Z J 1999 Genes Dev 13 284 294 12 Zandi E Rothwarf D M Delhase M Hayakawa M and Karin M X1997 Cell 91 243 252 13 Hu M C and Wang Y 1998 Gene Amst 222 31 40 14 DiDonato J A Hayakawa M Rothwarf D M Zandi E and Karin M 1997 Nature 388 548 554 15 Mercurio F Zhu H Murray B W Shevchenko A Bennet B L and Rao A 1997 Science 278 860 866 16 Rothwarf D M Zandi E Natoli G and KaringM 1 998 Nature 395 297 300 17 Yamaoka S Courtois G Bessia C Whiteside SIT Weil R Agou F Kirk H E Kay R J and Israel A 1998 Cell 93 1231 1240 18 Li Q Van Antwerp D
4. Mercurio F Lee K F and Yerma I M 1999 Science 284 321 32535 19 Li Z W Chu W Hu Y Delhase M Deernick T Ellisman M Johnson R and Karin M 1999 J Exp Med 189 1839 1845 20 Peters R T Liao S M 2000 Maniatis B MO Cell 5 513 522 Related Products IKKB Positive control Cat CY E1178 2 PRODUCED BY CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyelemco jp URL http wyww cyclex co jp CycLex CixcuLex 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 1178 14 Version 140318
5. Buffer provided 9 5 mL 950 PL 95 pL 20X ATP Solution 0 5 mL 50 pL 5 uL Total LOmL 1000 pL 100 pL You will need 80 90 uL of Kinase ReactiompBuffer per assay well Mix well Discard any unused Kinase Reaction Buffer afteruse 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 the foi pouch refold seal with tape and store at 4 C N Prepare all samples diluted with Kinase Buffer as needed All samples should be assayed in duplicate W To assay partially purifi d recombinant IKK add 10 uL of each fraction to the wells of the assay plate on ice Duplicate wells containing 10 m units 10 uL IKKP positive control Cat CY E1176 2 shouldbe ineluded 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 30 minutes 5 Wash wells fotr times with Wash Buffer making sure each well is filled completely Remove residual WashBuffer by gentle tapping or aspiration lon Pipette 100 UL of Anti Phospho IkBa S32 Monoclonal Antibody into each well cover with plate sealergor lid and incubate at room temperature ca 25 C for 30 minutes Discard any unused Cat CY 1178 6 Version 140318 IKK a and B Assay Inhibitor Screening Kit P 4 P ycLex User s Manual For Research Use Only Not f
6. CAUTION Sulfuri Acid is a strong acid Wear disposable gloves and eye protection when handling Stop Solution Cat CY 1178 5 Version 140318 IKK a and B Assay Inhibitor Screening Kit F 4 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product CycLex IKK a and 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 experimental conditions may_vary an aliquot of the IKKB Cat CY E1176 2 available separately from CycLex should be included th each assay as a positive control Disposable pipette tips and reagent troughs should be used for all liquid 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 O 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 viakof 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 100 uL at 20 C 3 Prepare Kinase Reaction Buffer by mixing following reagents 96 assays 10 assays 1 assay Kinase
7. Do not expose reagents t 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 required should be returned to the zip lock potiehy 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 in this kit may contain preservatives or other chemicals Care should be taken to avoid direct contact with these reagents Do not mouth pipet or ingest any of the reagents e Do not smoke eat or drink when performing theyassay or in areas where samples or reagents are handled e Dispose of tetra methylbenzidine TMB contdifiing 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 Solutionyand 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
8. IKKe activity involves incubatiomof the IKK sample with substrate either a natural or synthetic polypeptide such as IkBa S32 S36 peptide KKKERLLDDRHDSGLDSMKDEEYB in the presence of Mg and P labeled ATP he 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 radigactivitywon 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 Research Product CycLex KKee and p Assay Inhibitor Screening Kit uses a peroxidase coupled anti phospho IkBa S32 mongclonalgantibody as a reporter molecule in a 96 well ELISA format This assay provides a non isotopi Sensitive and specific method to detect IKK activity Cat CyY 1178 2 Version 140318 IKK a and B Assay Inhibitor Screening Kit F 4 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Principle of the Assay The CycLex Research Product CycLex IKK a and B Assay Inhibitor Screening Kit is a single site semi quantitative immunoassay for activities of IKKo and IKK Plates are pre coated with a substrate corresponding to recombinant IkBa which contains two serine residues that are phosphorylated by IKKa and IKK IKB kinases
9. 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 indicateahat 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 ih the CycLex Research Product CycLex IKK a and B 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 1178 10 Version 140318 4 IKK a and B Assay Inhibitor Screening Kit ye ex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Dose dependency of recombinant IKKB enzyme reaction 3 5 p 3 0 2 3 2 0 A 450 1 5 1 0 0 5 0 0 l l l J 0 5 10 15 20 IKK beta Positive control m units lt Fig 2 Time course of recombinant IKKB enzyme re
10. bottle containing 20 mL of the chromogenic substrate tetra methylbenzidine TMB Ready to use Stop Solution One bottle containing 20 mL of 1 N H2SO0 Ready to use Materials Required but not Provided e IKK positive control Available from CycLex IKKf positive control Cat CY E1178 2 IKKB enzyme Positive control should be added to the first well at 10 m units well Unused IKKB enzyme should be stored in aliquots at below 70 C 10X K252a 100 uM K252a is available from Wako Cat 1683 10 mM stock solution DMSO diluted 1 100 in Kinase Buffer e Pipettors 2 20 uL 20 200 uL and 200 1000 UL precision pipettors with disposable tips 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 e 500 or 1000 mL graduated ylinder e Reagent reservoirs Deionized watenof the highest quality e Disposable paper towels Cat CyY 1178 4 Version 140318 IKK a and B Assay Inhibitor Screening Kit F 4 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Precautions and Recommendations Store the ATP at 20 C in aliquots Store all other components at 4 C
11. en the IKK positive control 10 m units assay is included as an internal control for the phosphorylation reaction the absorbance value shouldbe greater than 1 0 with a background less than 0 2 2 For screening of purification chromatography fractions of recombinant IKK 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 IKK 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 IKK a and B Assay Inhibitor Screening Kit has been shown to detect the activity of IKK in column fractions of recombinant IKK Aihe assay shows good linearity of sample response The assay may be used to follow the purification offrecombinant IKK Troubleshooting 1 The IKK positive control should be run in duplicate using the protocol described in the Detailed Protocol Incubation times or temperatures signifi antly 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 an assay kinetics that are other than first order For a non linear curve point to point or quadratie curve fit methods should
12. is used to determine IKK a tiyity of off scale samples The readings at 405 nm should not replace the on scale readings at 450 nm Kinetic Assay 1 Remove the appropriate numb r of mi rotiter 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 w To assay partiallypurified recombinant IKK add 10 uL of each fraction to the wells of the assay plate on i e Duplicate wells containing 10 m units 10 uL IKKf positive control Cat CY E1176 2 shouldbe included in each assay as a positive control for phosphorylation 4 Begin kinase reaction by addition of 90 uL Kinase Reaction Buffer in duplicate per well in timed interyals suggested interval is 5 minutes but should be individually determined for each system After the fin li 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 1178 7 Version 140318 yclex User s Manual IKK a and B Assay Inhibitor Screening Kit 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 aspiratio
13. lowing table Although the level of A450 increases in Test sample when IKK enzyme activity is in the sample the high level OfpA450 is not observed in Inhibitor control ATP minus control and No enzyme control Ree reni Test Inhibitor ATP minus Positive No enzyme y reag Sample control control control control Kinase Reaction Buffer 90 uL 80 uL 9uL 90 uL Kinase Buffer provided 90 uL 10X K252a 100 uM 10 uL d Your enzyme fraction 10 uL 10 uL MuE IKK Positive Control 1 m unit uL 10 uL Buffer 10 uL 10X K252a 100 uM See page 4 section Materials Required but not Provided Cat CY E1176 2 See page 4 section Materials Required but not Provideds 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 on Buffer to each well and mixing thoroughly at room temperature Cover with plate sealer Incubate at 30 C for 30 minutes 2 Follow the Standard Assay steps 5 12 page 6 7 Cat CY 1178 9 Version 140318 IKK a and B Assay Inhibitor Screening Kit F 4 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Evaluation of Results 1 Average the absorbance values for the IKK sample duplicates positive control and all experimental sample duplicate values when applicable Wh
14. ls 4 Incubate for 30 min at 30 C Wash the wells Add 100 uL of Anti phospho kBa serine32 monoclonal antibody AS 2E8 In ubate for 30 min at room temp Washilthe wells Add 100 ub of HRP conjugated anti mouse IgG 4 Incubate for 30 min at room temp Wash the wells t Add 100 uL of Substrate Reagent t Add 100 uL of Stop Solution t Measure absorbance at 450 nm Cat CY 1178 3 Version 140318 IKK a and B Assay Inhibitor Screening Kit F 4 P 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 suppliedgand 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 afoil zip lock bag with a desiccant pack Wells are coated with recombinant IkBa as substrate of IKK 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 One vial of lyophilized ATP Na salt Anti Phospho IkBa 32 Monoclonal Antibody One vial containing 12 mE of anti phospho IkBa 32 monoclonal antibody AS 2E8 Ready to use HRP conjugated Anti mouse IgG One vial containing 12 mL of ARP horseradish peroxidase conjugated anti mouse IgG Ready to use Substrate Reagent One
15. n 7 Pipette 100 uL of Anti Phospho IkBa S32 Monoclonal Antibody into each well cover with plate sealer or lid and incubate at room temperature ca 25 C for 30 minutes Discard anyjunused antibody after use oo Wash wells five times as same as in step 6 9 Pipette 100 uL of HRP conjugated Anti mouse IgG into each well cover withgplate 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 room temperature for 10 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 spectrophotometrie plate reader at dual wavelengths of 450 540 nm Dual wavelengths of 450 550 or 450 595 nmcan 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 IKK 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
16. or NF B activation by all knownprofinflammatory stimuli including lipopolysaccharide LPS TNF and IL 1 18 19 Thus a selective inhibitor of IKKB would not only be of great interest as a potential anti inflammatory agent but alsof 8 a valuable tool to understand the mechanisms regulating NF B activation by these inflammatory agonists Peters et al 2000 reported the identification of a novel PMA inducible IKB Kinase complex They characterized one kinase from this complex which they designated IKKe 20 The IKKe protein shows 33 and 31 amino acid identity with IKKa and IKK respectively within the kinase domain and 27 amino acid identity with each throughout the entire sequence Although recombinant IKKe directly phosphorylates only Ser36 of IkBa the PMA activated endogenous IKK Jcomplex phosphorylates both critical serine Ser32 and Ser36 residues Remarkably this activity appears to be due to the presence of a distinct kinase in this complex A dominant negative mutant of IKKe Lys38 to Ala blocks induction of NF B by both PMA and activation of the T cell receptor buthas no effect on the activation of NF KB by TNF or IL1 These observations indicate that the activation of NF B requires multiple distinct IkB complexes that respond to both overlapping and discr te signaling pathways Measurement of IKK activity The protocol generally regarded as most sensitive for the quantitative measurement of IKKs including IKKa IKK and
17. or use in diagnostic procedures antibody after use 7 Wash wells five times as same as in step 5 8 Pipette 100 uL of HRP conjugated Anti mouse IgG into each well cover with plate sealer orJid and incubate at room temperature ca 25 C for 30 minutes Discard any unused cofjugate 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 uL of Stop Solution to each well in the same order as the pr viously added Substrate Reagent 12 Measure absorbance in each well using a spectrophotometric plate read r at dual wavelengths of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also bejused 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 t 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 val es do not exceed 0 25 units for the blank no enzyme control or 2 5 units for the XK positive control Note 3 If the microplate reader is not capable of readingyabsorbance greater than the absorbance of the IKK positive control perform a see d reading at 405 nm A new O D values measured at 405 nm
18. p IKK a and B Assay Inhibitor Screening Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Non Radioisotopic Kit for Measuring IKK activity CycLex IKK a and 6 Assay Inhibitor Screening Kit Cat CY 1178 Mmtended Use sccescieeetececteenisiiee eee 1 SOS yiee eee eE E EES 1 Tntroductio feen nnn e 2 Principle of the Assay 3 Materials Provided cccceeeseeeeeeeeeeees 4 Materials Required but not Provided 4 Precautions and Recommendation 5 Detailed Protocol cecceeccseeeseseseeeeeees 6 9 Evaluation of Results ccccceeeeeeeeeeeee 10 Assay Characteristics 10 Troubleshooting seeseeeeeeeeeeereerrerrerrrrreeee 10 Reagent SADLY savage sssissansndiaweteneeanssdasessbnaas 10 Example of Test Resilts cisicsssssssssletevnectenaees 14 13 References si cideisdicicssivveadis inition te o 14 Related Products cccccceececeeesseeeeeeeees 14 Intended Use The CycLex Research Product CycLex IKK o and p Assay Inhibitor Screening Kit designed to measure the activities of purified IKKa afd IKKf for the rapid and sensitive evaluation of inhibitors or activators The sequence specific phosphoserine monoclonal antibody used in this assay kit has been demonstrated to recognize the phospho serine 32 in recombinant IkBa which is efficiently phosphorylated by IKKs in vitro and in Vivo Applications of this kit include 1 Screening inhibitor
19. s or activators of IKKa and IKKB 2 Detecting the effects of pharmacological agents on IKKa and IKK activity This assay kit is for research use only and not for use in diagnostic or therapeutic procedures Storage e Upon receipt storeall components at 4 C e Don t expose reagents to excessive light Cat CyY 1178 1 Version 140318 IKK a and B Assay Inhibitor Screening Kit P 4 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Although a number of recent studies suggest that IxB degradation and nuclear translocation of NEacB may not be the sole regulatory events in the transcription of NF kB dependent genes 1 3 it hasbeen proposed that there is the central dogma of NF B activation which suggests that NF B is sequestered in the cytoplasm in resting cells by the inhibitory IkB proteins 4 7 In response to a variety of agonists IkB is rapidly phosphorylated ubiquitinated and degraded thus releasing NF B for translocation into the nucleus to initiate gene transcription 9 11 IkB kinase IKK is the convergence point in most signaling pathways activated by many stimuli leading to the inducible phosphorylation and degradation of IxB IKK is a multisubunit complex that contains two catalytic subunits IKKo and IKK and the regulatory subunit IKKy 8 12 17 Gene knock out studies have clearly demonstrated that IKKB and IKKy subunits of the IKK complex are required f
20. test chemicals cause an inhibitory effect on IKK activity the level of A450 is weakened as compared with Solvent control Assay reagents Test sample Solvent control Inhibitor control Kinase Reaction Buffer 80 uL 80 pL 80 uL 10X Inhibitor or equivalent 10 pL Solvent for Inhibitor 10 pL 10X K252a 100 0M 10 pL IKK Positive Control 1 m unit uL 10 uL 10 uL 10 pL or your enzyme fraction 10X K252a 1004M See page 4 section Materials Required but not Provided Cat CY Bl 176 2 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 IKK positive control to each well and mixing thoroughly at room Cat CY 1178 8 Version 140318 IKK a and B Assay Inhibitor Screening Kit F 4 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures temperature Cover with plate sealer Incubate at 30 C for 30 minutes 2 Follow the Standard Assay steps 5 12 page 6 7 Special considerations when measuring precise IKK activity In order to measure the activity of IKK correctly it is necessary to conduct the control experiment of Inhibitor control at least once for every experiment and ATP minus control at least one for the first experiment in addition to No enzyme control as indicated in the fol

CY-1178

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