#Bio rad western blot transfer protocol verification#

Due to the covalently bound compounds, proteins retain their fluorescence after activation and separation, thus providing the ability to verify protein transfer without any additional processing.ĥ.

#Bio rad western blot transfer protocol verification#

Alternatively, Bio-Rad's stain-free technology together with stain-free enabled imaging systems allows for instant verification of protein transfer in addition to increased sensitivity over Ponceau staining. Post-transfer staining is most commonly performed with Ponceau S, but this method involves time-consuming staining and destaining procedures and potentially introduces errors into western blotting results. A better method is to stain the membrane with a total protein stain that allows for the visualization of all proteins on the western blot and also allows the determination of transfer efficiency, molecular weight, relative quantities, and other properties of the transferred proteins. However, this method confirms only the transfer of the standards and does not provide a definitive confirmation for all the proteins on the western blot. Again, prestained standards can be used for visualization.

Bio-Rad's Trans-Blot ® Turbo™ System reduces transfer protocols to as little as 3 min while maintaining high-efficiency protein transfer across a wide range of molecular weights.Īfter proteins have been transferred, the transfer efficiency should be checked visually. Traditional semi-dry transfer systems, well suited for proteins ranging from 10–100 kD, can perform transfers in 15–60 min. Protocols utilizing standard wet or tank blotting systems typically require transfer times of one hour to overnight for a broad range of protein molecular weights. When activated by UV light, these compounds covalently bind to tryptophan residues in proteins and emit a fluorescent signal that is easily detectable with stain-free enabled systems such as Bio-Rad's ChemiDoc Touch, ChemiDoc MP, and Gel Doc EZ Imaging Systems.Īfter protein separation is verified, the transfer of proteins to the western blot membrane is most commonly performed by electrophoretic transfer. This stain-free technology is exclusive to Bio-Rad and is based on proprietary trihalo compounds. TGX Stain-Free Precast Gels contain unique compounds formulated into the gel chemistry that allow immediate visualization of proteins across the whole gel without staining. Another common method is the use of prestained standards, which does not give a complete picture of protein separation. However, this involves time-consuming staining and destaining procedures. SDS-PAGE followed by Coomassie staining is a standard, widely used method to visualize and confirm protein separation in the gel.

By comparison, complete electrophoretic separation can be performed at 300 V in 20 minutes with midi-format TGX gels. Typical sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) conditions for midi gels are 200 V for 30–45 minutes. The unique TGX gel formulation enables rapid separation of proteins at a high voltage while retaining Laemmli-like separation characteristics using standard sample and Tris/glycine running buffers. Bio-Rad offers TGX™ (Tris/glycine extended) and TGX Stain-Free™ Precast Gels for protein electrophoresis.