iBright™ Prestained Protein Ladder, 2 x 250 μL - FAQs

Most of the common gel running buffers are composed of Tris-glycine or Tris-tricine. Tris-glycine buffer systems are useful for separation of proteins over a wide range of molecular weights (5-300 kDa) and are compatible with denaturing or non-denaturing conditions. Tris-tricine buffer is generally recommended for the electrophoresis of low molecular weight proteins and peptides (<10 kDa) that need to be reduced and denatured prior to loading. Tris-acetate buffer system is used for separation of larger proteins (>200 kDa).

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The 2 unstained bands in the iBright Prestained Ladder contain an IgG binding site, allowing direct visualization with the same antibody-conjugate reagents used to detect the target protein. The proteins in the standard will not bind to IgM antibodies.

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Primary antibodies with low starting concentrations may result in insufficient chemiluminescent detection of the western blot positive control bands. If the unstained 30 kDa and 80 kDa bands produce weak or no signal, spike the diluted primary antibody with the corresponding rabbit IgG or mouse IgG to a concentration of 1-5 µg/mL, prior to secondary antibody incubation. Follow with respective secondary (GAM/GAR) incubation to increase the intensity of western blot positive control bands in the iBright Prestained Protein Ladder.

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If performing detection on a mini gel, the recommended loading volume is 1-3 µL. If performing visualization on a midi gel, 2-4 µL is recommended, and for detection, 2-3 µL is recommended. Optimal loading volume should be determined empirically.

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The iBright Prestained Protein Ladder has been optimized and designed for use with western blotting as it provides 2 control bands (unstained ladder bands) that are detected using the same antibody conjugate and protocol. The 2 unstained control bands contain IgG binding sites that can be visualized simultaneously with your target without any additional steps in the protocol. The 10 stained bands will appear during electrophoresis and transfer. These 10 bands will also appear in fluorescent imaging in the NIR channels. The 2 control bands will appear with chemiluminescent or fluorescent detection similar to your target.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.