Protein standards are mixtures of well-characterized natural or recombinant proteins that are loaded alongside protein samples in a gel. They are used to help monitor electrophoretic separation as well as estimate the size and concentration of the proteins separated in a gel. This section provides an overview of the different protein standards offered by Bio-Rad, their migration patterns and the range of molecular weight of proteins that can be used alongside these standards.
Related Topic: Protein Electrophoresis.
Select protein standards that offer:
Protein standards are available as prestained or unstained sets of purified natural or recombinant proteins. Prestained standards allow direct visualization of the proteins' migration during electrophoresis and are useful to assess their subsequent transfer to membranes. Prestained standards can be used for size estimation, however unstained protein standards are recommended for the most accurate size determination.
Bio-Rad offers a full range of prestained and unstained standards for various applications. Details of Bio-Rad's protein standards are provided in the tables and figures below.
Applications of Bio-Rad's protein standards.
MW = molecular weight. * Not to be confused with fluorescent total blot stains. Precision Plus Protein prestained standards contain dyes with fluorescent properties. See bulletin 5723 for details on using precision Plus Protein WesternC standards for fluorescent multiplexing. **Precision Plus Protein Standard Plugs are designed specifically for second dimension SDS-PAGE gels in 2-D experiments.
Bio-Rad's protein standards for western blotting applications.
Recombinant Precision Plus Protein Standards
Protein standards for western blotting applications selection guide.
* For colorimetric or chemiluminescence blot detection, StrepTactin-HRP or -AP conjugate is also needed.
Recombinant standards are engineered to display specific attributes such as evenly spaced molecular weights or affinity tags for easy detection on western blots. Bio-Rad's recombinant standards are the Precision Plus Protein™ standards family of products and are available in stained or unstained formats (see figure below). These standards contain highly purified recombinant proteins with molecular masses of 10–250 kD (or 2–250 kD for the Dual Xtra standards).
Unstained Standards Unstained standards contain only purified proteins, so they do not exhibit the variability in molecular weight sometimes observed with prestained standards. Therefore, unstained standards or standards with affinity tags for blot detection deliver high molecular weight recommended for the most accurate molecular weight determinations for gels or blots.
Overview of the StrepTactin detection system
Prestained Standards The ability to visualize prestained standards during electrophoresis makes them ideal for monitoring protein separation during SDS-PAGE. The proteins are labeled with fluorescent dyes, so can be used in fluorescence detection applications. The prestained markers remain visible on the membrane after transfer, which makes them useful for monitoring transfer efficiency and the general location of antigens in repetitive screening assays (Tsang et al. 1984). Bio-Rad offers both recombinant and natural prestained standards.
*Prestained standards are not recommended for use on gels that will be stained with fluorescent dyes.
Natural standards are blended from naturally occurring proteins. Though effective for monitoring gel separation and transfer efficiency, prestained natural standards have an inherent variability in the amount and location of dye that covalently binds to the protein. This may produce broader bands than seen with recombinant standards, making them less desirable for molecular weight estimations.
Bio-Rad offers the following prestained and unstained natural standards:
* Can also be used with other staining applications such as silver staining.
Bio-Rad's natural protein standards.
Tsang VC et al. (1984). Calibration of prestained protein molecular weight standards for use in the "Western" or enzyme-linked immunoelectrotransfer blot techniques. Anal Biochem 143, 304–307.
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