Determination of protein covalently bound to agarose supports using bicinchoninic acid.
A method using bicinchoninic acid (BCA) for the direct determination of protein covalently bound to agarose is described. The method involves the preparation of a standard curve using solubilized protein, the determination of the slurry concentration of the gel sample, the colorimetric assay of the gel slurry using BCA in a serum separator device, and the calculation of the amount of protein bound to the gel using the standard curve of the solubilized protein. The use of the BCA method for direct estimation of immobilized protein agrees well with the "balance" method which depends upon the measurement of protein depletion and yields highly reproducible results with a variety of coupling chemistries commonly used with agarose beads. The use of commercially available serum filters provides for a fast, less than 1 h, and convenient analytical format.
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T M
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Pierce Chemical Company, Rockford, Illinois 61105.
D003124:Colorimetry; D007202:Indicators and Reagents; D008722:Methods; D011506:Proteins / Q000032:analysis*; D011804:Quinolines; D012685:Sepharose; D012995:Solubility
D007202:Indicators and Reagents; D011506:Proteins; D011804:Quinolines; D012685:Sepharose; C047117:bicinchoninic acid