I ran out of Bromophenol Blue that is given in the standard recepie of Laemmli Buffer by CSH, now what concentration should I take Commassie Brilliant Blue G-250 as an alternative, should I take it the same concentration as Bromophenol Blue?
Coomassie Blue will bind to the protein, affecting its migration, so it is not appropriate for the sample buffer (except in blue native PAGE). You can run the gel without dye in the sample buffer. You can run the gel for a fixed amount of time based on experience or, for some gel setups, you can see the front by the change in refractive index across it. If you have prestained markers, you can also judge the end of the run based on the lowest marker. You can also substitute for bromphenol blue with about the same concentration of any other acidic, low molecular weight dye that does not interact with protein.
It is not applicable other than the Blue native page or tricine-PAGE application.
Here is a tip for the Tricine page app;
''You may use in your sample buffer; Coomassie Blue G and Phenol Red as tracking dyes instead of bromophenol blue. Coomassie Blue G gives a sharp dye front with Tricine SDS running buffers and migrates much closer to the moving ion front than bromophenol blue. Bromophenol blue runs more slowly than some peptides. This ensures that small peptides do not run off the gels.''
The concentration of Coomassie Brilliant Blue G-250 (CBB G-250) in Laemmli Buffer for SDS-PAGE can vary depending on the specific protocol and desired staining intensity. However, a commonly used concentration is around 0.025% to 0.05% (w/v). It's advisable to start with a lower concentration and optimize based on your specific needs. Keep in mind that the optimal concentration might also be affected by factors like the protein samples being analyzed and the type of gel system used.
- Badges
- Science topic
- Similar topics
- Filing