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Can these proteins be used for a functional assay with mouse blood?
Asked on Sep 20 2011
Thank you for contacting us. Activity of the proteins was assayed with a commercially available kit, as described below. (We do not offer the kit in our catalogue). The protein has not, to our knowledge, been used in any kind of functional assay of mouse blood but it may be possible to adapt the principle of the kit assay described below to your samples. Specific Activity: Assay is performed with specifications supplied with the Pefakit FXIII Incorporation Assay (Pentapharm LTD., Cat. # 501-00). Plasma based photometric determination of Factor XIII activity (See protocol included in the kit). Principle of the method - FXIII substrate fibrinogen (fbgn) is coated on the surface of a microtiter plate. Factor XIII in the sample is activated by thrombin and calcium ions (step omitted in the case of FXIIIa). In the incorporation step FXIIIa in the sample incorporates the substrate 5-biotinamidopentylamin (BAPA) into fbgn coated on the microtiter plate in the presence of calcium. The amount of incorporated BAPA is proportional to the FXIII activity of the sample. In the next step a strepavidine-alkaline phosphatase conjugate is bound to the incorporated BAPA. Alkaline phosphatase converts the synthetic substrate p-nitro phenyl phosphate into phosphate and p-nitrophenol which can be measured at 405 nm. Calculation of specific activity (Units/mg) - Results from the assay are reported as a percent (%) of NPP (based on controls provided by the assay kit). One unit is equal to the Factor XIII activity in 1 ml normal plasma (1 Unit = 100%). As an example 74.3% is equivalent to 0.74 U/ml. Divide U/ml by 101 to account for dilutions made in the standard curve. As in the example 0.74 U/ml / 101 = 0.0074 U/ml. Multiply this value by the dilution factor of the unknown sample (ex. 1:80,000) and divide by sample concentration (mg/ml). The result should be reported in Units/mg. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
Answered on Sep 20 2011