My sample buffer contains 50 mM Tris-HCl, pH 6.8, 2% SDS, 8 M urea, 10% glycerol, 2% mercaptoethanol and 0.02% bromophenol blue for getting the spore coat proteins. I think I can get rid of bromophenol blue because it just uses for visualize the proteins during gel electrophoresis. However, I wonder 2% SDS, 8 M urea, 10% glycerol, 2% mercaptoethanol whether can pass through FPLC column for protein separation and purification. Does anyone know about it?
Thanks both of you for giving the valuable comments. I think I will consider Superdex 75 or 200 for spore coat proteins separation. I don't think so I can include bromophenol blue because it will stain the columns. I concern about whether 2% mercaptoethanol and 10% glycerol can pass through the columns or not. It didn't mention in Superdex 75 and 200 data sheet.
Thanks Melissa :) I think I can omit 10% glycerol too since it is just used for increase the viscousity of the samples loading into gels. Am I right?
Ya, but the thing is I want to renature it back after denaturing by such high concentration of chaotropic agent (8 M urea), SDS and mercaptoethanol. Do you think the final samples I collect from FPLC is denature or native?
Thanks a lot! Do you think is it possible to renature my protein samples after running the columns?
I would reduce the urea to 6M to prevent the growth of urea crystals in your buffer reservoir., you do not need SDS (that is just for denaturing SDS/PAGE), and SDS will interfere with anion/cation exchange media (a good way to purify your protein). Gel filtration is used for concentrated samples that have contaminating proteins with very different MWs as compared to your protein. After you purify your protein in 6M urea you can use dialysis to renature it.
- Badges
- Science topic
- Similar topics
- Cell Biology
- Methods