The crosslink of a purified protein to NHS-activated magnetic beads works absolutely fine. But now I want to crosslink human skin tissue lysate to the beads and this does not work at all. The buffer does not contain amines (pure PBS). I personally assume that the high lipid content of the tissue lysate interferes with the esterification. Therefore, I need a quick and easy protocol for lipid extraction. I want to get rid of the lipids and purify the proteins from the skin tissue lysate. I tried two consecutive rounds of precipitation with acetone, but it didn't solve the problem. Any suggestions of how to get rid of lipids from skin tissue lysate?
In my opinion, the buffer can either promote or prevent enzyme-carrier or enzyme-enzyme interactions. The lipids remain associated with the proteins and intervenes during the conversion after immobilization
You could precipitate the protein with MeOH/CHCl3 (Wessel & Flügge 1984). For solubilisation of the resulting protein film you may need a detergent like SDS or Triton, but that shouldn't interfere with coupling.
Dear Hanen Ghamgui and Engelbert Buxbaum. I decided to use a different method, that does not require crosslink to magnetic beads. Anyway, thank you for your suggestions. Best regards, Thomas
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Proteins