TGX Stain-Free™ Gels
Stain-free gel chemistry uses a unique compound, which, when activated, reacts with tryptophan residues in the protein sample to emit a fluorescence signal. This allows the quick visualization of proteins without any staining steps. In addition, stain-free gel chemistry makes it possible to use total protein levels as a loading control rather than the housekeeping proteins used in traditional western blotting protocols. This negates the need to strip and reprobe blots and prevents any attendant errors that can be introduced at this step.
Fig. 1. Linearity comparison of stain-free total protein measurement and immunodetection of three housekeeping proteins in 10-50 µn;g of HeLa cell lysate.Bio-Rad’s TGX Stain-Free Gels use total protein normalization to produce a much greater linear dynamic range for measuring target protein levels (Figure 1). Housekeeping proteins such as β-actin, β-tubulin, or GAPDH are often very abundant in biological samples, which results in their signal being oversaturated compared to target proteins. Normalizing results to a total protein measurement corrects this problem, allowing a meaningful comparison even with low-abundance targets, and leads to far greater quantitative accuracy in measuring the levels of the protein of interest (Figure 2).
Bio-Rad’s V3 Western Workflow™ incorporates stain-free gel chemistry to streamline the western blotting protocol. This improved workflow, summarized below, saves time and increases accuracy and reliability throughout the western blotting process.
- Separate proteins using TGX Stain-Free Gels in as little as 15 min.
- Visualize protein separation in 1 min using the ChemiDoc™ Touch Imaging System.
Fig. 2. Comparison of kinase protein levels normalized by stain-free protein or actin loading controls. 10-50µn;g of HeLa cell lysate from the same sample were loaded onto a stain-free gel to probe MEK, Akt, and Erk. There should be no chnages in the kinase protein levels when data are normalized. Expected value (...)Transfer proteins efficiently and uniformly in 3 min using the Trans-Blot® Turbo™ Transfer System.- Verify transfer efficiency quickly using the ChemiDoc Touch Imaging System.
- Validate western blot data by normalization and analysis using the stain-free blot image as the total protein loading control.
