Protein assay methods

To determine the protein concentration, use filtrates from Sephadex G-25 (Micro-Spin) columns and proceed according to the protein assay method (BioRad). The results are given in mg protein/ml. 

In RBC, the activity for PTPS is calculated in pU/g Hb. The Hb concentration is determined in g Hb/ volume (ml) by standard laboratory methods for Hb. The results for tissues or other cells like fibroblasts or amniocytes are given in mg protein/ml. To determine the protein concentration, use filtrates from Sephadex G-25 (MicroSpin) columns and proceed according to the protein assay method (BioRad). 

This unit describes four of the most commonly used total protein assay methods. Three of the four are copper-based assays to quantitate total protein the Lowry method (see Basic Protocol 1 and Alternate Protocols 1 and 2), the bicinchoninic acid assay (BCA see Basic Protocol 2 and Alternate Protocols 3 and 4), and the biuret method (see Basic Protocol 3 and Alternate Protocol 5). The fourth is the Coomassie dye binding or Bradford assay (see Basic Protocol 4 and Alternate Protocols 6 and 7), which is included as a simple and sensitive assay, although it sometimes gives a variable response depending on how well or how poorly the protein binds the dye in acidic pH. A protein assay method should be chosen based on the sensitivity and accuracy of method as well as the condition of the sample to be analyzed. 

Sometimes the sample contains substances that make it incompatible with any of the protein assay methods. In those cases, some pretreatment of the sample is necessary. 

Each method has its advantages and disadvantages. No one method can be considered to be the ideal or best protein assay method. Because of this, most researchers keep more than one type of protein assay reagent available in their lab. 

The selection of a protein standard is potentially the greatest source of error in any protein assay. Of course, the best choice for a standard is a highly purified version of the predominate protein found in the samples. This is not always possible nor always necessary. In some cases, all that is needed is a rough estimate of the total protein concentration in the sample. For example, in the early stages of purifying a protein, identifying which fractions contain the most protein may be all that is required. If a highly purified version of the protein of interest is not available or it is too expensive to use as the standard, the alternative is to choose a protein that will produce a very similar color response curve with the selected protein assay method. 

For greatest accuracy of the estimates of the total protein concentration in unknown samples, it is essential to include a standard curve in each run. This is particularly true for the protein assay methods that produce nonlinear standard curves (e.g., Lowry method, Coomassie dye-binding method). The decision about the number of standards used to define the standard curve and the number of replicates to be done on each standard depends upon the degree of nonlinearity in the standard curve and the degree of accuracy required of the results. In general, fewer points are needed to construct a standard curve if the color response curve is linear. For assays done in test tubes, duplicates are sufficient however, triplicates are recommended for assays performed in microtiter plates due to the increased error associated with microtiter plates and microtiter plate readers. 

Table B1.1.5 demonstrates the relative degree of protein-to-protein variation that can be expected with the different protein assay methods. This differential may be a consideration in selecting a protein assay method, especially if the relative color response ratio of the protein in the samples is unknown. As expected, the protein assay methods that share the same basic chemistry show similar protein-to-protein variation.

The protein-to-protein variation observed with the various protein assay methods makes it obvious why the largest source of error for protein assays is the choice of protein for the standard curve. If the sample contained IgG as the major protein and BSA was used for the standard curve, the estimated total protein concentration of the sample will be inaccurate. Whether the concentration was underestimated or overestimated depends upon which total protein assay method was used. If the Coomassie... 

The amount of time required to complete a total protein assay will vary for the four colorimetric total protein assay methods described. For the puipose of providing an estimate of the amount of time required to perform a run by each method, it was assumed that the run included twenty samples and eight standards (including the blank) and that each sample or standard was assayed in duplicate using the standard tube protocol. The estimates do not include the time spent obtaining the samples or the time it takes to prepare the samples for... 

Peterson, G.L. 1977. A simplification of the protein assay method of Lowry, et al. Which is more generally applicable Anal. Biochem. 83 346-356. 

Sorenson, K. and Brodbeck, U. 1986. A sensitive protein assay method using micro-titer plates. Experientia 42 161 -162. 

Monitoring of solubility and dissolution Gravimetric analysis, a protein assay method (involving color reaction with bicinchoninic acid) and UV absorbance measurements at 214 nm Chemical analysis using TNBS reagent Fluorescence spectrophotometry... 

Table 1.1 summarizes detection limits and interferences for the five protein assay methods described here. 

TABLE 1.1. Detection Limits and Interferences for Protein Assay Methods... 

Li N, Li K A, Tong S Y. A novel protein assay method using tetraphenylpor-phin tetrasulfonate (TPPS4). Anal Lett 1995 28 1763-74. 

In the case of MPEG-DSPE liposome, amount of MPEG-DSPE incorporated was determined as by Alien et al. Briefly, the liposome dispersion was ultracentrifuged (150000 G, 20 min) to obtain a supernatant containing MPEG-DSPE free of liposomes. Then, the MPEG-DSPE concentration in the supernatant was determined by a Bio Rad Protein assay method. From this value, the amount of liposome-incorporated MPEG-DSPE was calculated. 

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