Benefits of Stain-Free Technology
Stain-Free technology is a unique in-gel chemistry that is available in Bio-Rad TGX Stain-Free precast gels. The gel formulation incorporates a trihalocompound that when exposed to UV irradiation activates a covalent reaction between the trihalocompound and tryptophan residues on the proteins in the gel, resulting in UV induced fluorescence.
Stain-Free technology enables fluorescent visualization of 1-D SDS PAGE gels and corresponding blots using Bio-Rad Laboratories’ (www.biorad.com) ChemiDoc MP imaging system (Figure 1A). The quality of SDS-PAGE separations before blotting can be easily monitored and the transfer efficiency of the blotting process can be quickly inspected by imaging both the membrane (Figure 1B) and the SDS gel after the blotting process. Furthermore, using Image Lab 4.0 software, which is included with the ChemiDoc MP, the relative amount of total protein in each lane on the blot can be calculated and used for quantitation normalization.
Stain-Free technology offers comparable sensitivity to conventional blot stains such as SYPRO Ruby and Ponceau S, and provides better reproducibility and linearity. Linear range of the Stain-Free technology is up to 80 µg protein for 18-well and up to 110 µg per lane for 12-well Criterion mid-size gels. This range fits well with usual protein loads in quantitative Western blotting experiments and enables loading control calculations over a wide protein-loading range.
HKP-based normalization with GAPDH, actin, or tubulin needs to be optimized for antibody dilutions, incubation times, and imaging settings. This is a very lengthy process compared to Stain-Free technology, which gives accurate, standardized protein-loading control with no optimization.