Protein concentrations, estimation

Estimation of Protein Concentrations in Solutions of Biological Origin... 

The WCE is clarified by two successive centrifugations at 16,100 for 2 and 10 min at 4°, respectively, using an Eppendorf F 45-24-11 rotor in a table centrifuge. After each centrifugation, the supernatant is carefully transferred to a new precooled Eppendorf tube, avoiding the lipid layer, and the total protein concentration (mg/ml) is estimated using the Bradford method (Biorad). 

Fig. 6. Determination of the critical protein concentration. (A) Plot of protein in the supernatant fluid after quantitatively sedimenting polymer from a polymerized solution of tubules and tubulin at steady state. The critical concentration, Ko, is determined from the value of the y axis intercept, and the fraction of active protein, y, from the slope. (B) The conventionally used experimental method for estimating the critical concentration. Note that the x axis intercept is actually Ko/y, instead of Kj,. Interpretation of the slope from such plots requires knowledge of the ratio of polymer weight concentradon to turbidity (given here as a). Data from experiments such as those in A may be used in conjunction with this plot to obtain the cridcal concentration, and this can serve as an internal test for self-consistency of the data.

Protein determination is necessary to estimate the amount of protein in the sample, to normalise against the protein concentration or during purification procedures. Depending on the amount of sample, accuracy and presence of interfering agents, one needs to decide on the method to be used. For accurate quantification, the sample protein is compared with a known amount of a standard protein which could either be the commonly used bovine serum albumin (BSA) or it could sometimes be immunoglobulin G (IgG). The various methods and their specifications are outlined below ... 

The amount of protein that is bound to the column can be estimated by subtracting the quantity of IgG that is eluted. This is only an estimate, but generally sufficient for antibody purification purposes. Continue with the subsequent steps if no more than 20% of the applied protein concentration is found in the eluate. Poly Prep columns are convenient since they are unbreakable, disposable, can be capped easily and securely at both ends, and have graduation markings for measuring column volumes. After the column has been packed, it should be stored at 4°C. Do not let the column warm up again or dry out, since this will introduce air bubbles which can cause protein denaturation. All subsequent purification steps should be performed at 4°C. 

The photometric estimation of protein concentration is subject to some special features Proteins interact with each other depending on their concentration and may change their secondary and/or tertiary structure in a concentration- dependent manner (especially denaturation in diluted solutions). These changes affect the absorption of light, i.e., concentration dependence of molar absorption coefficient e therefore, the Beer-Lambert law (eq. e) is not valid over a broad concentration range. 

This estimation of protein concentration is valid up to 20% (w/v) nucleic acid or an A280/A260 ratio < 0.6. 

In reproducing its own substance, the yeast cell produces an abundance of nucleic acids. Thus, not all the nitrogen in yeast is protein nitrogen, although calculation of protein concentration is based on this assumption. It is estimated that nucleoproteins make up 20 to 40% of bacterial nitrogen. 

Different proteins precipitate at different concentrations of an added salt. Hence, a fraction of proteins precipitating between two different concentrations of salt can be selected for further purification (Fig. 3-4). Protein concentrations can be estimated as described in Box 3-A. 

A series of reference proteins of known molecular masses are used to calibrate the column and Mr for an unknown protein is estimated from its position on the graph.195,196 Another modification of the method depends upon chromatography in a high concentration of the denaturing salt guanidinium chloride. The assumption is made that proteins are denatured into random coil conformations in this solvent.196... 

Although the spectrophotometric assay of proteins is fast, relatively sensitive, and requires only a small sample size, it is still only an estimate of protein concentration. It has certain advantages over the colorimetric assays in that most buffers and ammonium sulfate do not interfere and the procedure is nondestructive to protein samples. The spectrophotometric assay is particularly suited to the rapid measurement of protein elution from a chromatography column, where only protein concentration changes are required. 

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