Purification of Human Brain Tissue Factor
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Abstract:
Tissue factor (thromboplastin or Factor III), a glycoprotein cofactor, is required for Factor VII to express its catalytic activity, thereby initiating the extrinsic as well as intrinsic pathway of blood coagulation. Human brain tissue factor was purified 2,500-fold to 98% homogeneity from 2% Triton X-100 extraction of acetone dried brain powder with an overall yield of 36%. The method was based upon affinity chromatography utilizing the high affinity binding of tissue factor to Factor VII noncovalently complexed to immobilized anti-Factor VII-agarose beads. The apparent molecular weight of the purified tissue factor is 45,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its isoelectric point is 4.8-5.1 by column chromatofocussing and flat bed agarose isoelectric focussing.Keywords:
Agarose
Thromboplastin
Sodium dodecyl sulfate
Protein purification
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Agarose
Polyacrylamide
Color marker
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The isoelectric point of low temperature induced protein of straw mushroom was measured by isoelectric focusing electrophoresis with wide range pH3～10 and narrow range pH5～8 respectively.The isoelectric piont of the protein is 6.79,and the coefficients r of pH gradient curve is 0.937.
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Agarose
Orosomucoid
Polyacrylamide
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Abstract Polyacrylamide has been the matrix of choice for isoelectric focusing owing to the virtual absence of electroendosmosis in this medium. Certain inherent limitations associated with polyacrylamide have prompted some investigators to use low-electroendosmosis agarose for isoelectric focusing, but with limited success thus far. We have developed a method for isoelectric focusing in agarose for the classification of alpha 1-antitrypsin variants. Sera are applied directly to agarose gels containing a pH 4-5 ampholyte mixture, focused for less than 1 h, and directly immunofixed. Resolution of major bands is equivalent to polyacrylamide, and Pi M subtypes can be distinguished without the use of a separator. This application demonstrates the high resolution of isoelectric focusing in agarose, a more practical and convenient matrix than polyacrylamide.
Agarose
Polyacrylamide
Color marker
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