PROTEIN QUANTITATION

Quantitating the amount of protein present is critical for many applications. At Apollo Cytokine Research we aim to ensure that the amount of protein in our vials is as accurate as possible, thus delivering a product that customers can rely on.

A number of different protein quantitation methods are available. Assays that determine the total or specific protein content include the penultimate amino acid analysis (AAA); absorbance measurement at 280 nm; the Bradford and Lowry assays; the bicinchonic acid (BCA) and various fluorescence-based methods; enzyme-linked immunosorbent assays (ELISAs); and chip-based technologies such as the BIAcore. Estimates of concentration of pure proteins can be very inaccurate depending on the principle of the assay, unless the same pure protein is used as a standard.

Because different proteins have different amino acid compositions, the sensitivity and accuracy of colorimetric assays (Bradford, Lowry and BCA assays) for individual proteins may vary widely. For example, the Bradford assay is much more sensitive to immunoglobulin G (IgG) than to bovine serum albumin (BSA), so that if IgG is used as a standard the investigator is likely to underestimate the amount of protein in a sample. With BSA the investigator is likely to overestimate the amount. In addition, these assays are particularly vulnerable to the effects of common interfering agents such as detergents, reducing agents and urea. Recent advances have resulted in fluorescent-based assays that give higher sensitivity than the absorption-based methods. These assays however still use either BSA or IgG as a standard.

AAA entails the hydrolysis of the protein sample into amino acids, and the amount of each amino acid is determined empirically. This allows the calculation of the exact mass of the starting protein sample. While this is the only method that provides true protein quantitation, amino acid analysis does not work very well on glycosylated proteins as the sugar content forms a 'toffee like' substance and interferes with the hydrolysis of the protein. There is the additional constraint that specialized equipment and an experienced operator are required.

ELISAs and the Biacore assay rely upon the ability of the target protein to be recognized by an antibody or a functional binding protein. A drawback to these assays, in terms of quantitiating glycosylated proteins, is that most commercially available kits use antibodies raised against E. coli produced proteins. Recognition of native human proteins, with all their post-translational modifications, by these antibodies varies markedly (click here to view article on antibody recognition), resulting in a gross over- or underestimation of the protein concentration in human sera.

Measuring protein absorbance at 280 nm is a fast and convenient method, since no additional reagents or incubations are required. Most significantly, there is no requirement for a protein standard because the absorbance to protein concentration is linear, as described by the Beer-Lambert law[1]. Beer's Law states that molar absorptivity, also called molar extinction coefficient, is constant (and the absorption is proportional to concentration) for a given substance dissolved in a given solution and measured at a given wavelength. The molar absorbance coefficient of a polypeptide at 280 nm can be calculated from its amino acid composition, as only tryptophans, tyrosines and disulfide bonds absorb at 280 nm according to Gill and von Hippel[2,3]. Application of molar extinction coefficient in Beer's Law yields an expression of concentration in terms of molarity. However, the protein concentration in most protocols is provided in mg/ml rather than molarity. Therefore, at Apollo Cytokine Research we calculate the concentration factor based on the Beer-Lambert law, but we use the observed MW rather than the theoretical to convert from molarity to mg/ml as post-translational modifications such as carbohydrates in glycosylated proteins can add up to 50% to the theoretical mass (click here to view glycosylation summary). We are confident that these methods ensure the quantitation of our hcx^TM proteins is as accurate as possible.

General Reference: Stoscheck, CM. (1990) Quantitation of Protein. Methods in Enzymology 182, 50-69.
1. A = E x C x l, where A is the absorbance at 280 nm, E is the molar extinction coefficient, C the concentration in molarity and l the pathlength.
2. Gill, S.C. and von Hippel, P.H. (1989) Calculation of Protein Extinction Coefficients from Amino-Acid Sequence Data. Analytical Biochem. 182, 319-326.
3. This is the method used for calculation of Molar Extinction coefficients in the Analysis Tools on the ExPASy Server see ref. Protein Identification and Analysis Tools on the ExPASy Server; (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005). pp. 571-607

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• » Recombinant Human Proteins
• » ELISA Kits