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Molecular Weight Estimation and Quantitation of Protein Samples
Sponsored,vendor-submitted protocol   Sponsored by Bio-Rad Laboratories    Published in November 2007 (p.45)

Western blotting is a widely used and sensitive technique for the detection and characterization of proteins. This technique takes advantage of antibody recognition specificity toward an antigenic sequence in a target protein. In a typical Western blotting protocol, solubilized protein is separated by SDS-PAGE and electrophoretically transferred to a nitrocellulose or PVDF membrane (Figure 1).The choice of detection substrates and visualization methods can have a great impact on the quality of data from a Western blot.

Figure 1. (Click to enlarge)

Chemiluminescent detection using a digital imaging system offers important advantages over film-based or colorimetric procedures. A common objective of Western blot experiments is to obtain quantitative information about a target protein, including its molecular weight (MW) or concentration. For these applications, chemiluminescent images captured by digital documentation present a wider dynamic range and are easier to quantitate by densitometry, compared with colorimetric or film-based detection.

Protein standards play an integral part in analyzing Western blotting experiments. They can provide reliable MW sizing, and also serve as internal controls for monitoring an electrophoresis run and efficiency of blot transfer. Precision Plus Protein™ WesternC™ standards are prestained recombinant protein markers optimized for direct visualization in gels, transferred blots, and for parallel detection in chemiluminescent Western blotting applications (Figure 1).

The Immun-Star™ WesternC™ chemiluminescent detection kit contains a sensitive, chemiluminescent substrate for horseradish peroxidase (HRP) detection by digital imaging systems. The substrate is specifically designed to generate rapid, intense light emission of extended duration, which is optimal for use with charge-coupled device (CCD)-based imagers, such as the Molecular Imager® ChemiDoc™ XRS imaging system.

The Precision Plus Protein WesternC standards, StrepTactin-HRP conjugate, Immun-Star WesternC chemiluminescent detection kit, and the ChemiDoc XRS imager can be used to estimate the MW and amount (ng) of protein samples in Western blotting experiments. Blotted WesternC standards are used to graph log MW versus relative migration distance (Rf). The plot can then be used to calculate the MW of an unknown protein based on its Rf. In another application, a blotted dilution series of purified test protein of known concentration is used to construct a calibration curve of signal volume versus protein amount. The curve is then used to determine the amount of test protein in a crude sample.

StrepTactin is a trademark of Institut für Bioanalytick GmbH.

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