Sample buffer and protein assay - (Aug/01/2008 )
I have found a sample buffer by Sigma: (S3401 Sample Buffer Laemmli, 2x concentrate) with the formulation:
• 0.125 M Tris HCl, pH approx. 6.8
• 10% 2-mercaptoethanol (reducing agent)
• 4% SDS
• 20% glycerol
• 0.004% bromphenol blue
If you grow cells in a 10cm2 dish, would you put this on your samples, scrape off cells, put in an eppendorf tube and do a protein assay or do you use something else containing the protease inhibitors to harvest the cells?
Also I've read that you can't do a protein assay if bromophenol blue and reducing agents are present, so when do you do the protein assay?
I think I'm Ok with the Western Blot but I have no idea how to get my sample?
You would have to extract your cells of your dish with PBS or other Buffer, take a sample for protein analysis, and then add the loading buffer and boil. You might have to sonicate to get rid of the gloop, but it should be ok