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Protein C (PROC) ELISA Kit
|7 references available|
|Price||379.50 $ Plus shipping costs $45.00|
|Availability||Will be delivered in 2 to 3 Business Days|
|Method type||Sandwich ELISA|
|Alternative name||Protein C|
|Cross-Reactivity||Dog (Canine), Cow (Bovine), Monkey, Mouse (Murine), Rat (Rattus), Pig (Porcine), Rabbit|
|Sample Type||Cell Culture Supernatant|
|Sample Volume||50 µL|
|Assay Time||less than 4 hours|
|Components||Human Protein C Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human Protein C. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. Human Protein C Standard: Human Protein C in a buffered protein base (400 ng, lyophilized). Biotinylated Protein C Antibody (100x): A 100-fold concentrated biotinylated polyclonal antibody against Protein C (80µl). MIx Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml, 2 bottles). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (80µl). Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 ml).|
|Description||Protein C is a vitamin K-dependent plasma antithrombotic and anti-inflammatory zymogenic glycoprotein that is synthesized in the liver. Protein C has a light chain of 155 amino acids (21 kDa) and a heavy chain of 262 amino acids (41 kDa) linked by a disulfide bond. On endothelial cell membrane, thrombin-thrombomodulin complex cleaves a 12-reside peptide from protein C amino terminus of the heavy chain and converts it to activated protein C (APC). APC inactivates coagulation Factor Va and Factor VIIIa and performs a major role in regulating blood clotting, inflammation, and apoptosis (1-3). Protein C deficiency causes neonatal purpura fulminans, thrombophilia, and recurrent venous thrombosis (4-6). Protein C pathway components have been studied in the treatment of complex disorders, including severe sepsis, thrombosis, and ischemic stroke.|
|Minimum Detection Limit||3 ng/mL|
|Assay Precision||Intra-assay and inter-assay coefficients of variation were 4.8% and 7.3% respectively.|
|Principle||The AssayMax Human Protein C ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of human Protein C in urine, saliva, milk, and cell culture supernatant|
|Protocol||This assay employs a quantitative sandwich enzyme immunoassay technique that measures human Protein C in less than 4 hours. A polyclonal antibody specific for human Protein C has been pre-coated onto a 96-well microplate with removable strips. Protein C in standards and samples is sandwiched by the immobilized antibody and the biotinylated polyclonal antibody specific for Protein C, which is recognized by a streptavidin-peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.|
|Reagent Preparation||Freshly dilute all reagents and bring all reagents to room temperature before use. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. MIx Diluent Concentrate (10x): Dilute the MIx Diluent 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Standard Curve: Reconstitute the 400 ng of Protein C Standard with 2 ml of MIx Diluent to generate a solution of 200 ng/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting the standard solution (200 ng/ml) 1:2 with equal volume of MIx Diluent to produce 100, 50, 25, 12.5, 6.25 and 3.13 ng/ml solutions. MIx Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [Protein C] (ng/ml) P1 Standard (200 ng/ml) 200.00 P2 1 part P1 + 1 part MIx Diluent 100.00 P3 1 part P2 + 1 part MIx Diluent 50.00 P4 1 part P3 + 1 part MIx Diluent 25.00 P5 1 part P4 + 1 part MIx Diluent 12.50 P6 1 part P5 + 1 part MIx Diluent 6.25 P7 1 part P6 + 1 part MIx Diluent 3.13 P8 MIx Diluent 0.00 Biotin Protein C Antibody (100x): Spin down the antibody briefly and dilute the desired amount of the antibody 1:100 with MIx Diluent. Any remaining solution should be frozen at -20°C. Wash Buffer Concentrate (20x): Dilute the Wash Buffer Concentrate 1:20 with reagent grade water. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 1:100 with MIx Diluent. Any remaining solution should be frozen at -20°C.|
|Sample Collection||Urine: Collect urine using sample pot. Centrifuge samples at 800 x g for 10 minutes. Dilute samples 1:20 into MIx Diluent. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris. Collect supernatants and assay. Store samples at -20°C or below. Avoid repeated freeze-thaw cycles. Saliva: Collect saliva using sample tube. Centrifuge samples at 800 x g for 10 minutes. Dilute samples 1:800 into MIx Diluent. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Milk: Collect milk using sample tube. Centrifuge samples at 800 x g for 10 minutes. Dilute samples 1:6000 into MIx Diluent Store samples at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. 2|
|Assay Procedure||Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20 - 30 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccant inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 µL of Protein C standard or sample per well. Cover wells with a sealing tape and incubate for two hours. Start the timer after the last sample addition. Wash five times with 200 µL of Wash Buffer manually. Invert the plate each time and decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. If using a machine wash six times with 300 µL of Wash Buffer and then invert the plate, decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. Add 50 µL of Biotinylated Protein C Antibody to each well and incubate for one hour. Wash the microplate as described above. Add 50 µL of Streptavidin-Peroxidase Conjugate to each well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash the microplate as described above. 3 Add 50 µL of Chromogen Substrate per well and incubate for about 12 minutes or till the optimal blue color density develops. Gently tap plate to ensure thorough mixing and break the bubbles in the well with pipette tip. Add 50 µL of Stop Solution to each well. The color will change from blue to yellow. Read the absorbance on a microplate reader at a wavelength of 450 nm immediately. If wavelength correction is available, subtract readings at 570 nm from those at 450 nm to correct optical imperfections. Otherwise, read the plate at 450 nm only. Please note that some unstable black particles may be generated at high concentration points after stopping the reaction for about 10 minutes, which will reduce the readings.|
|Calculation of Results||Calculate the mean value of the triplicate readings for each standard and sample. To generate a standard curve, plot the graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance on the y-axis. The best-fit line can be determined by regression analysis using log-log or four-parameter logistic curve-fit. Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor. 3 Standard Curve The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.|
|Handling Advice||Prepare all reagents (working diluent buffer, wash buffer, standards, biotinylated- antibody, and SP conjugate) as instructed, prior to running the assay. Prepare all samples prior to running the assay. The dilution factors for the samples are suggested in this protocol. However, the user should determine the optimal dilution factor. Spin down the SP conjugate vial and the biotinylated-antibody vial before opening and using contents. The kit should not be used beyond the expiration date.|
|Material not included||Microplate reader capable of measuring absorbance at 450 nm. Pipettes (1-20 µL, 20-200 µL, 200-1000µLand multiple channel). Deionized or distilled reagent grade water|
|Storage Comment||Store components of the kit at 2-8°C or -20°C upon arrival up to the expiration date. Store SP Conjugate and Biotinylated Antibody at -20°C Store Microplate, Diluent Concentrate (10x), Wash Buffer, Stop Solution, and Chromogen Substrate at 2-8°C Opened unused microplate wells may be returned to the foil pouch with the desiccant packs. Reseal along zip-seal. May be stored for up to 1 month in a vacuum desiccator. Diluent (1x) may be stored for up to 1 month at 2-8°C. Store Standard at 2-8°C before reconstituting with Diluent and at -20°C after reconstituting with Diluent.|
|Research Area||Cardiovascular, Coagulation|
|Restrictions||For Research Use only|
Kisiel, Ericsson, Davie: "Proteolytic activation of protein C from bovine plasma." in: Biochemistry, Vol. 15, Issue 22, pp. 4893-900, 1977 (PubMed).
Foster, Yoshitake, Davie: "The nucleotide sequence of the gene for human protein C." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 82, Issue 14, pp. 4673-7, 1985 (PubMed).
Esmon, Owen: "Identification of an endothelial cell cofactor for thrombin-catalyzed activation of protein C." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 78, Issue 4, pp. 2249-52, 1981 (PubMed).
Witt, Beck, Seydewitz et al.: "A novel homozygous missense mutation (Val 325-->Ala) in the protein C gene causing neonatal purpura fulminans." in: Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis, Vol. 5, Issue 4, pp. 651-3, 1995 (PubMed).
Dahlbäck, Carlsson, Svensson: "Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 90, Issue 3, pp. 1004-8, 1993 (PubMed).
Hansson, Sörbo, Eriksson: "Recurrent venous thromboembolism after deep vein thrombosis: incidence and risk factors." in: Archives of internal medicine, Vol. 160, Issue 6, pp. 769-74, 2000 (PubMed).
Mosnier, Zlokovic, Griffin: "The cytoprotective protein C pathway." in: Blood, Vol. 109, Issue 8, pp. 3161-72, 2007 (PubMed).