How to make a neat gel for master thesis? - Expressed protein on SDS-PAGe gel (Apr/29/2008 )
I am expressing a protein in E.coli and want to show the process (ie. uninduced, induced, pellet, crude extract and pure protein) on a gel photo. But, how do I know how diluted I must make the sample with denaturing sample buffer and how much to load of each to make a nice gel?
I would measure protein content in each sample and load somewhere between 10 and 100 ug per well (for 10x10cmx1mm gel). For most of my samples 20-30 ug per well works fine.
Thank you for the quick reply!
But isn't it difficult to measure protein conc in the culture pellets? (uninduced and induced culture is just centrifuged and the pellet is to be resuspended in sample buffer)
If you resuspend the pellet from 1ml of culture in 500microliters of Sample buffer 10 to 15 microliters is OK not to saturate the commassie gel, adjust your volume to this You might need to shear the DNA with a small needle to avoid the blub of genomic DNA which might alter the migration this depends very much of the type of E Coli strain and the extraction process