Protein sticking to membrane - Protein concentration woes (Apr/29/2005 )

During my purification, I have noticed substantial loss of protein from my very first column. Initially I attributed this to the protein aggregation facilitated by the presence of my his-tag, but I have no remedied this problem (thx to this forum! ). However, after my most recent prep and doing a Bradford, I STILL noticed significant protein loss.

After much thinking, I am now convinced that it is due to the nature of my protein and it sticking to the amicon YM-10 membrane. My protein binds to negatively charged species and the membrane, I was once told is negatively charged.

My question is, is there any way I can get around this? I need to have this protein concentrated for the crystallographers. I am going to try lyophilizing it, however, my protein is quite finicky and I am not sure how that will work out. Any suggestions? I previously used centricons and I believe the membrane is the same as those of the amicon ones.

Suggestions would be great. I lost over 15 mg of protein!!!!!

Thanks!

It is seem that your protein is highly hydrophobic, I would suggest to wash the membrnaes with 0.1 M carbonate buffer pH 11 to disrubt such interactions. this buffer is used to remove prepheral membrane proteins for the membrane by disrubting hydrophobic interactions of the protein with the plasma membrane. good Luck!