So, I have a recombinant his tagged protein being expressed in e coli. So, we grow a culture of E coli, spin it down and separate the cells from the media/supernatant. The cells are lysed and the lysate run over a nickel column. Purification is not an issue, but the analysis of the proteins is.
When I mix the lysate or the Nickel column flow through with 4xSDS loading buffer (commercial) we end up getting some kind of precipitate. We initially thought, DNA crashing out. So we thought, a clean up is necessary.
We performed TCA precipitation (followed by washes with acetone). We tried resuspending the pellet in 4xSDS loading buffer, and we have the same issue - the protein will simply not dissolve. So what we thought earlier to be DNA crashing out, is actually protein that doesn't seem to like 4xSDS.
So, we added some elution buffer (nickel resin elution - tris, imidazole and salt based at pH 8.0) to the mix to see if we could dissolve it - but it remains as a precipitate.
Have you observed anything similar where the ppt refuses to dissolve in the 4xSDS after TCA precipitation? If so, how did you get around it?