Protein concentration, determination

Figure 1. Top Turbidity, measured at 350 nm, as a function of microtubule polymer mass concentration (expressed as mg/mL polymerized tubulin). Tubulin solutions of varying concentrations were polymerized until they reached stable plateau values in a Cary 118C spectrophotometer. Each sample was then transferred to an ultracentrifuge tube, and microtubules were pelleted, separated from the unpolymerized tubulin in the supernatant fraction, and then resuspended for protein concentration determination. The corresponding turbidity and polymer mass concentrations are plotted here. Bottom Time-course of tubulin polymerization assayed by turbidity.Repro-duced from MacNeal and Purich with permission from the American Society for Biochemistry and Molecular Biology.

Dilute sarcolemmal vesicles from Protocol 5.3.2 to about 1 mg pro-tein/ml with Soln. A (for protein concentration determination the Lowry method is recommended). Add 1 pi of Soln. B per 100 pi SL dilution, vortex and put on ice. Pipet the probes in triplicates into Eppendorf tubes according to Table 5.6. Label the ouabain-containing tubes with those without ouabain should be signed... 

Lumbar spinal cord sections were homogenized individually in ice-cold phosphate buffer saline (PBS) containing a cocktail of protease inhibitors (Sigma, code P8340) and centrifuged at 14,000g for 10 min at 4 °C. The supernatant was then collected and protein concentration determined using the Bio-Rad DC protein assay (Bio-Rad Laboratories, Milan, Italy). 

For elution, protein concentration determination, and storage steps, follow the same procedure as explained in Subheading 3.2 (Steps 10-12). 

Use lysis buffer if needed to adjust for equal loading among the samples according to the protein concentrations determined in the Bradford assay. 

The Lowry assay [16] uses the reaction of cupric sulfate at alkaline pH in the presence of tartrate, producing a blue chromogen formed from four peptide bonds and one atom of copper. Addition of folin phenol reagent further enhances the color, with a maximum absorbance at 750 nm. The Lowry assay demonstrates the greatest sensitivity of the common protein concentration determination methods and varies only slightly when using the two common calibrators, BS A and BGG. Not surprisingly, this remains a very commonly used method. 

With so many choices, it is important to be aware of the advantages and limitations of these methods. The following case study illustrates the importance of appropriate protein concentration determination methodology. 

If more DTNB is used, the excess aromatic thiol or disulfide could compete with the protein for Hg(II) and thus expose additional protein thiols for reaction with the DTNB reagent. Protein thiol titrations with DTNB are often subject to inaccuracies resulting from oxidation of the thiol residues, background DTNB hydrolysis, and the inherent uncertainty in measuring protein concentrations (161). Any or all of these factors may play a role in the discrepancies in results described above. In efforts to eliminate these contributions, we performed DTNB measurements with relatively low DTNB concentrations of O.OOlAf and at relatively low pH to eliminate hydrolysis interference. Protein concentration determinations were based on the experimentally determined extinction coefficient of 58(X) M cm found for MerR (145). 

It may be conceivable that the structure formation of barnacle adhesive is determined by critical self-assembly concentrations of the adhesive proteins within an interfacial gap between a barnacle base and a substrate. It can further be suggested that the biopolymers form coherent gel structures, in which two transitions of critical protein concentrations determine the arrangement of adhesive globules from a dense sheet-like formation to a slightly loose sponge-like formation to a very loose branched or web-like structure. 

Figure 8.7. Plasmon absorbance of gold NPs in absence and presence of proteins (concentration determination is described in Section 8.5). Measurements were performed immediately after mixing the protein and gold solutions.

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