Globulin Correction Factors

The greatest problem is rinsing. Proposals have been made to modify the original Grassmann method, for instance, by the use of a Soxhlet(G3), but the only substantial technical improvement is described by Ooster-huis (02) who uses 10 liters of an aqueous solution of 1 % acetic acid for washing 10 paper strips, with a 2-hour washing time (D3). 

Bromophenol blue, suggested by Durrum (D13) and Jencks (J3), is used as an aqueous solution of 0.1 g per liter containing 5 % acetic acid and 5 % zinc sulfate. Here, dye fixation affects considerably the protein factor. Heat must be applied according to the standard schedule of 30 minutes at 120°C (D15). 

Some authors, however, prefer an alcoholic solution of the dye, containing mercuric chloride as fixing agent (K22). 

Rinsing is first done with an aqueous solution of 2 % acetic acid and finally with an acetate buffer at pH 3.6, since the influence of pH on this dye is considerable (D15). 

Azocarmine B was originally used by Turba and Enenkel (T4) as a saturated solution and is prepared in a mixture of 50 % methanol, 10 % glacial acetic acid, and 40 % water. Rinsing is done with methanol and later on with 1 % acetic acid. The wavelength needed for colorimetry is 520m[x. Protein factors again vary widely 1 (T4), 1.35 (M3), 1.5 (V2), 1.6 (E4, E5), 1.8 (P18). 

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A second possibility is to divide the fractions into the albumin on one side and the globulins on the other and to apply one single correcting factor to the albumin. This factor is a compound of the protein and of the paper factor. Protein and paper errors are most important for the large and concentrated albumin fractions. Fortunately they partly compensate each other, the paper error tending to lower the albumin value and the protein error tending to raise it. We use 1.5 in our own experimental setup and multiply the amido black-albumin surface by this figure. 

In order to overcome the relative error in albumin and globulin estimations due to the higher binding capacity of albumin, correction factors of 1.6 to 2.4 have been suggested (P3). However, for some dyes (e.g., nigrosin) the difference in binding capacity to serum albumin and globulin is not constant at different protein concentrations (Sll). 

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