How to Choose the Ideal Protein Standard

April 23, 2012 at 2:16 pm 1 comment

In the following post, we will share with you a practical guide for selecting appropriate protein standards. The information was distilled from an article published in issue 127 of Bio-Rad Laboratories’ BioRadiations Magazine and can be found in its entirety on page 11 when clicking on this link. The guide will be broken down into both gel electrophoresis and western blotting applications.

Gel Electrophoresis
Unstained Standards
When using gel electrophoresis for molecular weight (MW) determination, unstained standards are the ladders of choice. Unstained standards are unencumbered by protein dyes and therefore give the most accurate electrophoretic mobility pattern. They are also required when a fluorescent dye is used to stain the gel or blot to visualize total protein.

MW estimation can be determined by plotting unknowns agains a standard curve which is generated by calculating the relative mobility of each protein standard and plotting the mobility of each band against the log10 of its MW.

Prestained standards
Prestained standards are ideal for monitoring the progress of electrophoresis.

Western Blotting
In western blotting, prestained standards are the standards of choice since they can be used for both MW estimation as well as for determining the efficiency of protein transfer. This holds true for both colorometric and chemiluminescent protein (or protein tag) visualization. However, when using a fluorescent-based stain for visualization, an unstained standard must be used.

All in one
In addition to the “usual” protein standards, Bio-Rad Laboratories manufactures the Precision Plus Protein WesternC standards which contains a Strep-tag affinity peptide that has intrisic binding affinity for both native streptavidin or StrepTactin and can be “lit” up with the appropriate chemiluminescent stubstrate during your western blot chemiluminescent development. This is a very cool tool since it allows you to use an “all in one” standard for monitoring the progress of electrophoresis, protein transfer efficiency and protein standard visualization on the actual film (or chemidoc image).

Fluorescent Standards
For those who are utilizing fluorescently labeled antibodies in your experiments, Bio-Rad’s Precision Plus protein standards, dual color, Kaleidoscope and WesternC prestained standards include bands that fluoresce when excited by different wavelengths of light which can be useful for multiple detection when multicolored antibodies are chosen.

Entry filed under: Bio-Rad Product Highlight, Bio-Rad Tech Note, Proteomic Technotes. Tags: , , .

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