cGMP Direct Immunoassay Kit (Colorimetric)
Contents
1. Factors Cytokines amp Chemokines e Antibodies to Apoptosis amp Cell Signaling Molecules Metabolism Assay Kits e Cholesterol Assay Kit HDL LDL assay kit Glucose Assay Kit Lactate Assay Kit Pyruvate Assay Kit NAD P H NAD P Assay Kit Glutathione Assay Kit Nitric Oxide Detection Kit Tel 408 493 1800 Fax 408 493 1801 www biovision com tech biovision com Page 2 of 22. Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA For research use only rev 08 14 e To determine whether interference is presence in your sample you may make two different dilutions If the two different dilutions of sample show good correlation in the final calculated cGMP concentrations purification is not required If you do not see good correlation of the different dilutions deproteinize the sample by using TCA or 10 Kd molecular cut off microcentrifuge filters e Some organic solvents may interfere with the assay and may need to be removed prior to the assay C Sample Preparation Urine Plasma and Culture Media Urine plasma and culture media may be tested directly after adding 1 10 volume of 1M HCl and remove precipitates if occur Culture Cells For suspension cells collect by centrifugation Add 1 ml of 0 1M HCI for every 35 cm of surface area e g 10 cm plate at 70 confluency is 110 cm so use 3 1 ml Incubate at room temperature for 20 minutes on ice For adherent cells add the HCL directly scrape cells off the surface Dissociate sample by pipetting up and down until suspension is homogeneous Transfer to a centrifuge tube and centrifuge at top speed for 10 min The supernatant can be assayed directly Protein concentration gt 1 mg ml is recommended for reproducible results Tissue Samples Cyclic nucleotides may be metabolized quickly in tissue so it is important to rapidly freeze tissues a
3. BioVision cGMP Direct Immunoassay Kit Colorimetric Catalog K372 100 100 assays Store at 20 C Introduction Adenosine and guanosine 3 5 cyclic monophosphate cAMP and cGMP are important second messengers involved in many physiological processes BioVision s CGMP Direct Immunoassay Kit provides a direct competitive immunoassay for sensitive and quantitative determination of cGMP level in biological samples The kit utilizes the recombinant Protein G coated plate to anchor cGMP polyclonal antibody cGMP HRP conjugates directly competes with cGMP from samples for binding to the cGMP specific antibody on the plate After incubation and washing the amount of cGMP HRP bound to plate can easily be determined by reading ODaso nm The intensity of OD4so nm is inversely proportional to the concentration of cGMP in samples The kit provides a new acetylation procedure that improves detection signal significantly The kit can detect 0 04 10 pmol well 0 008 2 uM cGMP samples Kit Contents Component K372 100 Color Code Storage Part 100 assays Cap Color Temperature Number 10X cGMP Assay Buffer WM K372 100 1 Standard cGMP 10 nmol Yellow K372 100 2 Neutralizing Buffer NM K372 100 3 Acetylating Reagent A Violet K372 100 4 Acetylating Reagent B Black K372 100 5 Anti cGMP pAb BSA Red K372 100 6 cGMP HRP BSA Green i K372 100 7 HRP Developer Amber K372 100 8 Protein G Coated Plate 6522 1 cGMP Assay Protocol A Re
4. agent Preparations e Dilute the 10X cGMP Assay Buffer to 1X Assay Buffer with MilliQ water Store at 4 C e Reconstitute the Standard cGMP pellet may not be visible in 1 ml of 0 1M HCI not provided vertex for 10 seconds to generate 10 pmol l cGMP stock standard solution e Reconstitute rabbit anti cGMP pAb and cGMP HRP each with 1 1 ml of the 1X Assay Buffer as stock solutions e Unused well strips can be kept at 20 C with the desiccants stable for up to 1 month e The kit should be stored at 20 C After reconstitution some components may be stored at 4 C as instructed above stable for up to 1 2 months e NOTE Acetylating Reagent B is very volatile and hence the vial has to be tightly capped and stored only at 4 C B General Consideration e cGMP samples in 0 1 M HCI final concentration is stable and can be used directly in the assay Make dilutions of your sample with 0 1 M HCI to the range of 0 04 10 pmol well 0 008 2 uM e Plasma serum whole blood and tissue homogenates often contain phosphodiesterases and large amount of immunoglobulins lgs which may interfere with the assay However preparing samples in 0 1 M HCI can generally inactivate phosphodiesterases and lower the concentration of Igs making the samples suitable for the assay Both phospho diesterases and lgs can also be removed by 5 TCA precipitation or by using 10 Kd molecular weight cut off microcentrifuge filters BioVision Cat 1997 25 BioVision
5. ay Mix 1 volume of Acetylating Reagent A Violet cap with two volumes of Acetylating Reagent B Black cap in a microtube Prepare just enough for the experiment Use within 1 hour 8 Add 5 ul of the Acetylating Reagent Mix directly into each test solution both standard and sample IMMEDIATELY vortex 2 3 seconds following each addition without delay one tube at a time and incubate at room temperature for 10 min 9 Add 845 ul 1X Assay Buffer into each tube mix well Use for below quantification Note The acetylation step improves the assy sensitivity significantly and avoid the interferences of many components in unpurified samples If cGMP in your samples are very low the acetylation reagents can be dried after step 8 without dilution step 9 to minimize the volume Then reconstituted in a 50 100 ul volume of Assay Buffer Tel 408 493 1800 Fax 408 493 1801 www biovision com tech biovision com Page 1 of 2 BioVision er For research use only Quantification cGMP RELATED PRODUCTS 1 Add 50 ul of the acetylated Standard cGMP and test samples from Step 9 above to each well of the Protein G coated 96 well plate We suggest duplicate assays for each sample Apoptosis Detection Kits amp Reagents e Annexin V Kits amp Bulk Reagents e Caspase Assay Kits amp Reagents and standard i e Mitochondrial Apoptosis Kits amp Reagents 2 Add 10 ul of the reconstituted cGMP antibody per well to the standard cGMP and sample e Ap
6. fter collection e g using liquid nitrogen Weigh the frozen tissue and add 5 10 volume of 0 1M HCI Homogenize the sample on ice using a Polytron type homogenizer Spin at top speed for 5 min and collect the supernatant The supernatant may be assayed directly CGMP Assay Protocol Prepare cGMP Standard Curve and Samples 1 Add 200 ul of the 10 pmol ul standard cGMP stock into 800 ul of 0 1M HCI to generate 2 pmol yl cGMP working solution The diluted cGMP should be used within 1 hour 2 Label 11 microcentrifuge tubes 10 5 2 5 1 25 0 625 0 3125 0 156 0 078 0 039 0 0_B pmol 50 ul Note these concentrations represent what will finally be in the wells after the dilutions mentioned below 3 Add 200 ul of the 2 pmol ul cGMP into the tube labeled 10 pmol enough for 20 tests add100 ul 0 1M HCI into the rest of tubes 4 Transfer 100 ul from the 10 pmol tube into the labeled 5 pmol tube mix Continue the serial dilution by transferring 100 ul from the 5 pmol tube to 2 5 1 25 0 625 0 3125 0 156 0 078 0 039 pmol tubes Discard 100 ul from the 0 039 pmol tube The diluted cGMP should be used within 1 hour 5 Label new tubes for test samples add 100 ul each test sample per tube We suggest using different dilutions for each sample dilute with 0 1M HCl 6 Add 50 ul of Neutralizing Buffer to each tube to neutralize the HCI in the samples and standards 7 Prepare Acetylating Reagent Mix Note 5 ul is needed for each ass
7. optotic DNA Ladder Detection Kits wells except the well with 0_B pmol cGMP Note Do not add cGMP antibody into the well e TUNEL based DNA Fragmentation Assay Kits with 0_B pmol cGMP instead add 10 ul of 1X Assay Buffer for background reading e Apoptosis Inducers and Set Incubate for 1 hour at room temperature with gentle agitation Cell Fractionation System OOPS Note Using a repeating pipette is recommended for minimizing pippetting errors 7 e a Toe e Kit 3 Add 10 ul of CGMP HRP to each well and incubate for 1 hr at room temperature with gentle enoa e Shasta A E Kii agitation e Cytosol Particulate Rapid Separation Kit 4 Wash 5 times with 200 ul 1X Assay Buffer each time Completely empty the wells by e Mammalian Cell Extraction Kit tapping the plate on a fresh paper towel after each wash step e FractionPREP Fractionation System Add 100 ul of HRP developer and develop for 1 hour at room temperature with agitation Cell ee Sil a Assay Kit Stop the reaction by adding 100 wl of 1M HCI not provided to each well sample color e Senescence Detection Kit should change from blue to yellow e High Throughput Apoptosis Cell Viability Assay Kits 7 Read sample at OD 450 nm e LDH Cytotoxicity Assay Kit 8 Subtract OD450 nm background reading the well with 0_B pmol cGMP from all samples e Bioluminescence Cytotoxicity Assay Kit and standards Plot standard curve to observe the linear portion then replot only the linear e Live Dead Cell Staining Ki
8. t portion and in Excel add a trendline then use the trend line linear formula y mx b Cell Damage amp Repair Calculate amount of cGMP in samples after correcting the for dilution factors e HDAC Fluorometric amp Colorimetric Assays amp Drug Discovery Kits 9 Calculations e HAT Colorimetric Assay Kit amp Reagents C Sa Sv pmol l or nmol m or uM Where Sa is the cGMP amount pmol from the Standard Curve Sv is the sample volume ul added into the assay wells after dilution factor correction 1 6 1 2 0 8 OD 450 nm 0 4 0 0 mn 0 039 1 56 6 25 12 5 25 100 cGMP pmol well cGMP Standard Curve The assay was performed following the kit protocol FOR RESEARCH USE ONLY Not to be used on humans BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA e DNA Damage Quantification Kit e Glutathione amp Nitric Oxide Fluorometric amp Colorimetric Assay Kits Signal Transduction e cAMP amp cGMP Assay Kits e Akt amp JNK Activity Assay Kits e Beta Secretase Activity Assay Kit Obesity Research e Recombinant Adiponectin Survivin amp Leptin e CETP amp PLTP Activity Assay amp Drug Discovery Kits e Cholesterol and HDL LDL Quantification Kits e Fatty acid and Triglyceride Quantification Kits e NAD P NAD P H and ADP ATP Assay Kits Antibodies amp Recombinant Proteins many e Adiponectin Proteins amp Antibodies e C5a Recombinant Protein e Protein G amp Protein A Bulk Quantity e Growth
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