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CY-1161

1. Assay reagents Test sample Solvent Inhibitor control control cGMP plus Kinase Reaction buffer 80 pL 80 pL 80 pL 10X Inhibitor or equivalent 10 uL Solvent for Inhibitor 10 pL 10X K252a 10 pM 10 pL Cyclex cGK positive control 0 2 units uL 10 uL 10 uL 10 uL or your enzyme fraction 10X K252a See page 4 section Materials Required but not Provided Cat CY E116141 and Cat CY E1161 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 gt uL Jof Diluted cGK positive control 0 2 units uL to each well and mixing thoroughly at r 6om temperature Cover with plate sealer and incubate at 30 C for 30 minutes 2 Follow the Standard Assay steps 5 10 page 7 Cat CY 1161 8 Version 140318 yclex User s Manual Special considerations when measuring cGK activity In order to measure the activity of cGK family correctly it is necessary to conduct the control experiment of Test Sample cGMP minus or 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 and Positive control as indicated in the following table Although the level of A450 is several times higher in Test sample cGMP plus than in Test sample cGMP minus wh
2. yclex Detailed Protocol User s Manual Cyclic GMP dependent protein kinase cGK Assay Kit For Research Use Only Not for use in diagnostic procedures The CycLex cGK Assay Kit is provided with removable strips of wells so the assay can be carriedgout on separate occasions using only the number of strips required for the particular determination Since experimental conditions may vary an aliquot of the cGK I positive control Cat CY E1161 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 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 O 0X ATP provided lyophilized Mix gently until dissolved The final concentration of the 20X A TP Solution should be 2 5 mM Store the solution in small aliquots e g 100 uL at 20 C 3 Prepare cGMP plus Kinase Reaction Buffer by mixing following reagents 96 assays TO assays Kinase Buffer provided 9 5 mL 950 uL 20X ATP Solution 0 5 mB 50uL 5 mM cGMP 20 uL 2 uL Total 40 02m 1002 uL You will need 80 90 uL of cGMP plus Kinase Reaction Buffer per assay well Mix well Disc
3. For Research Use Only Not for use in diagnostic procedures Evaluation of Results 1 Average the absorbance values for the cGK sample duplicates positive control and all experimental sample duplicate values when applicable When the cGK positive control 2 units assay is included as an internal control for the phosphorylation reaction the absorbance value should be greater than 0 with a background less than 0 15 2 For screening of purification chromatography fractions plot the mean absorbance valuessfor each of the samples on the Y axis versus the fraction number on the X axis to determine the location of the eluted purified cGK 3 For kinetic analysis plot the mean absorbance values for each of the time points Onhe Y axis versus the time of each reaction minutes on the X axis Assay Characteristics The CycLex Research Product CycLex cGK Assay Kit has been showntto detect the activity of cGK family in column fractions The assay shows good linearity of sample f sponse The assay may be used to follow the purification of cGK containing fractions or may be aised to detect the presence of cGK activity in cell lysates Troubleshooting 1 The cGK standard should be run in duplicate usingiithe 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 are of
4. in diagnostic procedures References Pfeifer A Aszodi A Seidler U Ruth P Hofmann F and Fassler R Intestinal secretory defeets and dwarfism in mice lacking cGMP dependent protein kinase II Science 274 2082 2084 1996 2 Tamura N Itoh H Ogawa Y Nakagawa O Harada M Chun T H Suga S Yoshimasa T and Nakao K cDNA cloning and gene expression of human type IJ alpha cGMP dependent protein kinase Hypertension 27 552 557 1996 3 Orstavik S Natarajan V Tasken K Jahnsen T and Sandberg M Characterization of the human gene encoding the type I alpha and type I beta cGMP dependent protein kinase PRKG1 Genomics 42 311 318 1997 4 Li Z Xi X Gu M Feil R Ye R D Eigenthaler M Hofmann F and Du dA stimulatory role for cGMP dependent protein kinase in platelet activation Cell 112 77 86 2003 5 Yamahara K Itoh H Chun TH Ogawa Y Yamashita J Sawada N Fukunaga Y Sone M Yurugi Kobayashi T Miyashita K Tsujimoto H Kook H Feil R Garbers DL Hofmann F Nakao K Significance and therapeutic potential of the natriuretic peptid s cCGMP cGMP dependent protein kinase pathway in vascular regeneration Proc Natl Acad Sci 7 S A 100 6 3404 9 2003 Related Products Cyclic GMP dependent protein kinase cGK Positive Control Catalytic domain Cat CY E1161 1 Cyclic GMP dependent protein kinase cGK Positive Control Full length Cat CY E1161 2 An
5. purification methods can be used to isolate cGKs The following protogols 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 cGKs 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 crudejextract cell lysate or sample fraction taken prior to a purification step One eight well strip of the substrate plate should be sufficient for this initial experiment All sample preparations should be performed at 4 C and recovered fractions be kept at 4 C to prevent loss of enzymatic activity CAUSION It should be noted that this assay kit detects not only cGK activity but also other protein kinases in crude extract and column sample You should trace cGK protein level by western blotting in column fractions Column Purification Fractions 1 Homogenize fresh 10 15 g of tissues lung cerebellum etc in threegyolumies of extraction buffer 50 mM potassium phosphate buffer pH 7 0 1 mM EDTA 1 mM EGTA 0 2 mM PMSF 1 ug mL pepstatin 0 5 ug mL leupeptin 5 mM beta glycerophosphate mM_ NaF 2 mM Na3VO 10 mM beta mercaptoethanol in a Potter Elvehjem tissue homogenize 2 Centrifuge th
6. the presence of Mg and ATP The amount of phosphorylated substrate is measured by binding tt with a horseradish peroxidase conjugate of 10H11 a anti phospho G kinase substrate threonine 68419 sp cific 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 quantified by spectrophotometry and reflects the relative amount of cGK actiyity in the sample For kinetic analysis the sample containing cGK activity is added to the wells in a similar fashion and at varying times the reaction is stopped by the addition of a chelator sodium ethylenediamine tetraacetate EDTA and the amount of phosphorylated substrate determined as before The CycLex Research Product CycLex cGK Assay Kit is designed to accurately determine the presence and relative amount of cGK activity in tissue cytosols celk extracts purification column fractions and for the non isotopic kinetic analysis of cGK activity Careful attention to extraction methods and the assay protocol will provide the investigator With areliable tool for the evaluation of cGKs Summary of Procedure Add 100 uL of sample to the wells 4 Incubate for 30 min at 30 C Wash the wells t Add 100 uL of HRPxconjugated anti phospho specific antibody 4 Incubate for 1hr at room temp Wash the wells t Add 100 uL of Substrate Reagent t Add 100 uL of
7. 5 nm A new O D values measured at 405 nm is used to determine cGK activity 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 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 N Prepare all samples 4diluted with Kinase Buffer as needed All samples should be assayed in duplicate w To assay individualColumn fractions add 10 uL of each fraction to the wells of the assay plate on ice Duplicate wells containing 10 uL of cGK positive control Cat CY E1161 should be included in each assay aS apositive control for phosphorylation 4 Begin kinas r action by addition of 90 uL cGMP plus Kinase Reaction Buffer in duplicate per w ll in timediintervals 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 Cat CY 1161 7 Version 140318 yclex oo Cyclic GMP dependent protein kinase cGK Assay Kit User s Manual For Research Use Only Not for use in diagnostic procedures addition of 150 uL 0 1 M Na EDTA pH 8 0 to each well Wash wells five times with Wash Buffer making sure each well is filled completely Remove r
8. Stop Solution t Measure absorbance at 450 nm Cat CY 1161 3 Version 140318 Cyclic GMP dependent protein kinase cGK Assay 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 suppliedand are sufficient for one 96 well microtiter plate kit Microplate One microplate supplied ready to use with 96 wells 12 strips of 8 wells in afforl zip lock bag with a desiccant pack Wells are coated with recombinant G kinase 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 One vial of lyophilized ATP Na salt HRP conjugated Detection Antibody One vial containing 12 mL of MRP horseradish peroxidase conjugated anti phospho G kinase substrate 10H11 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 H SO Ready to use Materials Required but not Provided e cGK positive control 1 Catalytic domain cGK positive control 2 Full length Available from CycLex Cat CY E1161 1 and Cat CY E1161 2 respectively One vial contains 4000 units 100 uL cGK enzyme Positive control s
9. T cell by 8 CPT cGMP and SNP 1 1 4 1 2 1 0 0 8 lt 0 6 0 4 0 2 0 0 E ATP ATP None 100uM 100uM SNP 1 8 CPT cGMP Fig 7 Inhibition of cGK activity that is stimulated wittu M GMP by Rp 8 CPT cGMP in vitro 120 100 80 60 40 20 Relative intensity control 0 Cat CY 1161 0 10 50 100 Rp 8 CPT cGMP uM 15 Version 140318 Cyclic GMP dependent protein kinase cGK Assay Kit Pa P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 8 RESOURCE Q column elution profile of cGK cGK enriched fraction concentrated isoelectric point precipitation from rabbit brain extract 18 C e cGMP L6 O cGMP 1 4 Nacl2 1 2 1 0 0 8 NaCl2 conc mM 0 6 cGK Activity A 450 0 4 0 2 0 0 0 10 20 30 40 50 Fraction Number A Fig 9 Superdex 200 elution profile of cGK cGK e d fraction separated by RESOURCE Q column Fr 27 32 A 1 8 300 250 0GMP 150 A280 mAU cGK Activity A450 0 3 50 0 10 20 30 40 50 7 8 9 Fraction Number C CY 1161 16 Version 140318 Cyclic GMP dependent protein kinase cGK Assay Kit F 4 T ycLex User s Manual For Research Use Only Not for use
10. ard any unused cGMP plus Kinase Reaction Buffer fter use 4 Prepare Kinase Reaction Buffer by mixing following reagents When you conduct several control experiment described in page 9 96 assays 10 assays 1 assay Kinase Buffer provided 9 5 mL 950 uL 95 pL 20X ATP Solution 0 5 mL 50 pL 5 uL Total 10 mL 1000 uL 100 uL Standard Assay 1 Remove the appropriate number of microtiter wells from the foil pouch and place them into the well holder Retufn 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 T assay individual column fractions add 10 uL of each fraction to the wells of the assay plate on ice Crude lysates or cell extracts should be added to wells either neat or diluted as described above with Kinase Buffer if necessary Suggested starting dilutions are 1 2 1 5 and 1 10 Duplicate wells containing 10 uL of cGK positive control Cat CY E1161 should be included in each assay Cat CY 1161 6 Version 140318 Cyclic GMP dependent protein kinase cGK Assay Kit P 4 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures as a positive control for phosphorylation 4 Begin the kinase reaction by addition of 90 uL of cGMP plus Kinase Reaction Buffer per well cover with plate sealer and incubate at 30 C fo
11. d the cell pellet with an appropriate extraction buffer for example 20 mM Tris pH 7 4 150 mM NaCl 1 mM EDTA 1 mM EGTA 0 2 mM PMSF 1 pg mL pepstatin 0 5 ug mL leupeptin 2 mM NaF 0 2 mM Na3VO4 5 mM beta mercaptoethanol and lyse the resuspended cells usingyeither a Dounce Homogenizer sonication or three cycles of freezing and thawing 3 Transfer extracts to microcentrifuge tubes and centrifuge for 5 minutes 4 Aliquot cleared lysate to a clean microfuge tube These samples are now ready fopfanalysis according to the instructions provided in the Detailed Protocol NOTE THE ABOVE PROCEDURES ARE INTENDED ONLY AS A GUIDELINE THE OPTIMAL EXPERIMENTAL CONDITIONS WILL VARY DEPENDING ON THE PARAMETERS BEING INVESTIGATED AND MUST BEQDETERMINED BY THE INDIVIDUAL USER NO WARRANTY OR GUARANTEE OF PERFORMANCE USING THESE PROCEDURES IS MADE OR IMPLIED Example of Test Results Fig l Dose dependency of cGK 2 8 2 8 2 6 H e cGMP 2 6 2 4 M e cGMP 2 4 2 2 29 2 0 2 0 1 8 1 8 1 R y Z a SS ul i 1 2 lt 1 2 1 0 1 0 0 8 0 8 0 6 0 6 0 4 0 4 0 2 0 2 0 0 0 0 0 0 0 5 1 0 1 5 2 0 0 0 0 5 1 0 1 5 2 0 cGK Ia full length units cGKIa catalytic domain units Cat CY 1161 12 Version 140318 Fag Cyclic GMP dependent protein kinase cGK Assay Kit Ce YCLEX User s Manual For Research U
12. e All microplate strips that are not immediately required should be returned to the zip lock peuch 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 of 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 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 whenNecessary Biological samples may be contamimated 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 1161 5 Version 140318
13. e homogenate for 20 min at 30 000 x g to pellet the insoluble membrane organelle fraction 3 Dilute clear lysates by adding 4 volumes of ic cold 5 mM potassium phosphate buffer pH 7 0 10 mM beta mercaptoethanol 4 Adjust the pH of the lysates solution to 5 4 by_slow addition of 0 5 N acetic acid with stirring 5 After stirring for 10 min centrifuge the solution for 15 min at 30 000 x g to obtain the precipitate which contains most of cGK enzyme actiVity 6 Dissolve the precipitate in 5 ml of DE buffier 20 mM Tris HCl pH 7 5 60 mM NaCl 0 5 mM EDTA 1 mM EGTA 0 2 mM PMSF 1 wg mBypepstatin 0 5 ug mL leupeptin 2 mM NaF 0 2 mM Na3VOu 10 mM beta mercaptoethanol 7 Remove the precipitate by centrifuge the solution for 15 min at 30 000 x g 8 Apply the supernatant t0a 1 x 8 cm column of DEAE cellulose Whatman DE 52 equilibrated with DE buffer 9 Wash the colummngwith five column volumes of DE buffer 10 Elute the column With a linear gradient of 0 06 0 3 M NaCl total six column volumes in DE buffer collecting 1 2 mL fractions These samples are now ready for analysis according to the instructions proyided inthe Detailed Protocol Cat CY 1161 11 Version 140318 Cyclic GMP dependent protein kinase cGK Assay Kit Pe P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Cell Culture Lysates 1 Harvest and pellet cells by centrifugation using standard methods 2 Resuspen
14. en cGK family enzymedictivVity 1s in the sample the high level of A450 is not observed in Inhibitor control ATP minus control and No enzyme control Cyclic GMP dependent protein kinase cGK Assay Kit For Research Use Only Not for use in diagnostic procedures Adcavieanauis Test Sample Test Sample Inhibitor ATP minus Positive No enzyme y reag cGMP plus cGMP minus control control control control cGMP plus Kinase Reaction buffer 90 uL 80 uL 90 uL 90 uL Kinase Reaction buffer 90 uL Kinase Buffer provided 90 uL 10X K252a 10 uM 10 uL Your enzyme fraction 10 uL 10 uL 10 uL WuL CycLex cGK positive control 0 2 unit pL i Wyk Buffer 10 uL 10X K252a See page 4 section Materials Required but not Provide Cat CY E1161 1 and Cat CY E1161 2 See page 4 section MaterialsRequired but not Provided 1 Following the above table add the Reagents tojeach well of the microplate Finally initiate the reaction by adding 10 uL of Your enzyme fraction or Diluted cGK positive control or Buffer to each well and mixing thoroughly at room temperature Cover with plate sealer and incubate 30 C for 30 minutes 2 Follow the Standard Assay steps 5 l0ppagey7 Cat CY 1161 Version 140318 Cyclic GMP dependent protein kinase cGK Assay Kit F 4 P ycLex User s Manual
15. esidual Wash Buffer by gentle tapping or aspiration Pipette 100 uL of HRP conjugated Detection Antibody into each well cover with afplate sealer and incubate at room temperature ca 25 C for 60 minutes Discard any unused conjugate jafter use Wash wells same as in Step 6 Add 100 uL of Substrate Reagent to each well and incubate at room temperature ca 25 C 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 r ader 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 of inhibitors In order to estimate the inhibitory effect on cGK family 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 n addition to Test sample as indicated in the following table When test chemicals cause an inhibitory effect on cGK 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 2
16. hould be added tothe first well at 2 units well e cyclic GMP guanosine 3 5 monophosphatesis available from Sigma Cat G 6129 Make 5 mM stock solution e Rp 8 pCPT cGMPS Rp 8 para chlorophenylthio guanosine 3 5 monophosphate is available from Calbiochem Cat 370677 e 10X K252a 10 uM K252a is available ffrom Calbiochem Cat 420297 1 mM stock solution DMSO diluted 1 100 in Kinase Burffer e Pipettors 2 20 uL 20 200 uL and 2001000 uL precision pipettors with disposable tips e Precision repeating pipettor e Wash bottle or multichannel ispenser for plate washing e Microcentrifuge and tubes forsample preparation e Vortex mixer e Plate reader capable of measuring absorbance in 96 well plates at dual wavelengths of 450 nm 540 nm Dual wavelengthspof 450 550 or 450 595 nm can also be used The plate can also be read at a single wavelength of 450 am which will give a somewhat higher reading e 500 or 1000 mL graduated cylinder e Reagent reservoirs Deionized waterof high quality Cat CY 1161 4 Version 140318 Cyclic GMP dependent protein kinase cGK Assay 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 Do not expose reagents to excessive light Avoid freeze thaw cycles e Allow all the components to come to room temperature before use
17. istinct genes Smooth muscle platelets and cerebellum contain high concentrations of eGK I whereas cGK II is highly concentrated in brain lung and intestinal mucosa The function of cGK II is not well understood although there is evidence that it mediates intestinal secr trompof water and electrolytes induced by the E coli toxin STa and the intestinal peptide guanylin Activation of cyclic GMP dependent protein kinase cGK is an important eyent in the regulation of blood pressure and platelet function Upstream signals include the generation ofiitric oxide NO by NO synthases and the subsequent rise in cGMP levels mediated by NO dependent guanyl cyclases GCs The identification of new cGK activators by high throughput screening HTS may lead to the development of a novel class of therapeutics for the treatment of cardiovascular diseases Measurement of cGK activity The protocol generally regarded as most sensitive for the quantitativesmeasurement of cGK activity involves incubation of the cGK sample with substrate either a natural orsynthetic polypeptide such as Histone H1 substrate peptide in the presence of Mg and Ptabeled ATP The reaction is terminated by spotting a sample onto a filter paper disc followed by immersion in acid to precipitate the radiolabeled product The filter papers are then washed extensively to remove unincorporated radiolabel and the radioactivity is counted While sensitive this method is labor intensi
18. mily members Additionally column fractions of any cultured primary cell cell line or tissue homogenate can be assayed for cGK family activity with the CycLex Research Product CycLexcGKAssay Kit if the appropriate dose of cGK specific inhibitor e g Rp 8 pCPT cGMPS Applications of this kit include 1 Monitoring the purification of cGK 2 Screening inhibitors or activators of cGK 3 Detecting the effects of pharmacological agents on cGK This assay kit issfor research use only and not for use in diagnostic or therapeutic procedures Storage e Upon receipt store all components at 4 C e Dont expose reagents to excessive light Cat CY 1161 1 Version 140318 Cyclic GMP dependent protein kinase cGK Assay Kit P 4 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Nitric oxide NO and a broad spectrum of hormones drugs and toxins raise intracellular cGMP concentrations and thereby regulate a great variety of functions including smooth muscle relaxation neuronal excitability and epithelial electrolyte transport Pfeifer et al 1996 noted that dependingson the tissue an increase in cGMP concentration leads to the activation of different receptors such asicyclic nucleotide phosphodiesterases The identification of the physiologic mediator of cGMP is complicated by the existence of 2 forms of cGMP dependent protein kinase cGK types I and II which are encoded by d
19. p Cyclic GMP dependent protein kinase cGK Assay Kit A ed c ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Non Radioisotopic Kit for Measuring cGK Activity CycLex Cyclic GMP dependent protein kinase cGK Assay Kit Cat CY 1161 Intended Use sssssccccsissessssesectessssessteiaccesscsees 1 SOE ATT 1 Introduction srusen 2 Principle of the Assay 3 Materials Provided c c ccccceeesssseeeeseeees 4 Materials Required but not Provided 4 Precautions and Recommendation 5 Detailed Protocol ccccccccccssssseeeceeeeeeees 6 9 Evaluation of Results cccccccceeeeesseees 10 Assay Characteristics s 10 Troubleshooting ss ccsscasnisswssaseceatsesnaaindensvevnnes 10 Reagent Stability scscccadesssandasvenasenccenaasarsarsents 10 Sample Preparation 11 42 Example of Test Resaltsccsccccccisssastivensseaceasaes 12 16 Referens ssssiseccicdisdivses eriein ak N Related Products cccccccccccssssseeeeeeesenees 17 Intended Use The CycLex Research Product CycLex Cyelie GMP dependent protein kinase cGK Assay Kit is primarily designed to measure the activities of purified the cGK family of kinases for the rapid and sensitive evaluation of inhibitors or activators The phospho specific monoclonal antibody used in this assay kit has been demonstrated toerecognize the phospho threonine 68 119 residues on G kinase substrate which is phosphorylated by GK fa
20. r 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 6 Pipette 100 uL of HRP conjugated Detection Antibody into each well cover with a plat sealer and incubate at room temperature ca 25 C for 60 minutes Discard any unused conjugate 7 Wash wells five times as same as in step 5 oo 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 spectrophotometri plate reader at dual wavelengths of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can alsopbe 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 eSsentialto 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 cGK positive control Note 3 If the microplate reader is not capablegOfweading absorbance greater than the absorbance of the Weel positive control perform a seconddeading at 40
21. se Only Not for use in diagnostic procedures Fig 2 Time course of cGK kinase reaction 2 0 1 8 1 6 1 4 1 2 1 0 0 8 0 6 0 4 0 2 0 0 e ATP ATP Q A 450 0 20 40 60 80 100 120 Reaction Time min Ww Fig 3 Km for ATP 4 500 4 000 3 500 3 000 2 500 2 000 1 500 1 000 500 y 39 231x 200 65 R 0 9937 Km 5 1 uM v s A450 min uM 0 20 40 60 80 100 s ATP uM C CY 1161 13 Version 140318 Fag Cyclic GMP dependent protein kinase cGK Assay Kit Ce ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 4 Effect of broad spectrum kinase inhibitor K252a on cGK I alpha activity IC50 0 1 uM Relative Activity control a D oo Ws 0 01 0 10 1 00 10 00 K252a uM vy y cGMP in Vitro Fig 5 Activation of full length cGK I alpha expresse Ww S D S S 5 Nn S Nn Relative Activity control 0 uM cGMP 0 0 001 0 010 0 100 1 000 10 000 cGMP concentration uM C CY 1161 14 Version 140318 Yyelex Cyclic GMP dependent protein kinase cGK Assay Kit User s Manual For Research Use Only Not for use in diagnostic procedures Fig 6 Activation of full length cGK I alpha expressed in 293
22. the first order Variations in the protocol can lead to non linearity of li ycurve as can assay kinetics that are 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 D not allow the plate to dry out Add Substrate Reagent immediately after wash Reagent Stability All of the reagents ineluded in the CycLex Research Product CycLex cGK Assay 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 and cGK positive control Cat CY E1 161 must be stored at below 70 C Coated assay plates should be stored in the original foil bag sealed by the zip lock and containing a desiccant pack For researchwse only not for use in diagnostic or therapeutic procedures Cat CY 1161 10 Version 140318 Cyclic GMP dependent protein kinase cGK Assay Kit F 4 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Sample Preparation Numerous extraction and
23. ti phospho G substrate T68 119 monoclonal antibody 10H 11 Cat CY M1017 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 wwyneyclex co jp CycLex Circulsex 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 1161 17 Version 140318
24. ve 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 cGK Assay Kit uses a peroxidase coupled anti phospho G kinase substrate threonine 68 119 monoclonal antibody as a reporter molecule in a 96 well ELISA format This assay provid s a non isotopic sensitive and specific method to measure cGK family activity Cat CY 1161 2 Version 140318 Cyclic GMP dependent protein kinase cGK Assay Kit P 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 cGK Assay Kit is a single site semi quantitative immunoassay for cGK activity Plates are pre coated with a substrate corresponding to recombinant G kinase substrate which contains threonine residues that can be phosphorylated by cGK family members including cGKI and cGKII The detector antibody specifically reacts with only the phosphorylated form of threonine 68 119 residues on cGK substrate The CycLex cGK Assay Kit may be used to determine the pr sence of GK activity in cell lysates purification column fractions or to follow the kinetics of a purified or partially purified cGK protein as well as screening cGK inhibitors To perform the test the sample is diluted in Kinase Buffer pipetted into the wells and allowed to phosphorylate the bound substfate in

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