How to deal with insoluble proteins? - (Jul/03/2008 )
I want to compare the ratio of soluble protein against insoluble protein. But I have problem on finding the concentration of insoluble protein and preparing SDS-PAGE sample. Can anyone help me?
you can use this protein assay on the insoluble proteins after you have solubilized them with sds loading buffer without the bromphenol blue (add that after taking the sample for determination):
pierce bca protein assay for use with reducing agent
We normalise the OD of cells from the start. Take your culture and measure the OD. Resuspend the cells such that the OD is some multiple of this (we'd often use 10-20x because it helped keep the volumes manageable, others pick an OD and run with that). Once you have resuspended the cells and lysed them and spun out the debris, resuspend the pellet in the same volume as the soluble fraction. This way, you approximate the concentrations of soluble and insoluble proteins.