I am trying to purify a protein that is MBP tagged. This protein, is known in literature to be insoluble. I have employed the pMAL-C2x vectore for the same. It has a lac inducible promoter. Now, I did a protein profile of the cells upon induction and expected a 98kDA band to show up(45 kDa MBP + 53 kDa protein). It did not however, so I'm trying to check if it precipitated down owing to its insolubility. I pelleted down the entire 5ml of culture and stored in -20 for later use. Now, the pellet wont resuspend in LB, nor will it pellet completely upon centrifugation. I need very little pellet to work. Any solution?
Use a sterile loop and directly suspend the pallet in LB manually. Sometimes the pallet sticks strongly to the bottom. Then you can vortex to mix it uniformly.
Just checking but did you add glycerol or any form of antifreeze before freezing the cells or were you freezing the cells only for protein purification later
You can add 0.1N NaOH+1%SDS mixture (Depending on how much protein or OD you want to load). Let me know if it works. Best Luck.
- Badges
- Science topic
- Similar topics
- Chemistry
- Sulfites
- Inorganic Chemistry
- Inorganic Chemicals
- Salts