I recently performed protein extraction with RIPA buffer of some cells I collected. The intended purpose was to use it to load in an SDS PAGE gel for subsequent Western Blot. I made the mistake of lysing the cells in too much RIPA buffer and although there is protein, the sample is too diluted. I want to load 20 micrograms of total protein per lane, and I have roughly 400 microlitres of a protein solution at 0.13 microgram/microlitre. Since there is no gel that supports having 150 microlitres of sample loaded into a lane, I am looking for a way to concentrate what I have rather than just discarding it.
My postdoc once made a gel comb with one big deep tooth, then poured a custom 2-part gel using the usual formula. By running at low voltage at first, the protein concentrates at the stacking/running interface.
Hello Krish Dodani
You may use acetone precipitation to precipitate the proteins. Add at least three volumes of ice-cold acetone to the lysate. Allow proteins to precipitate at -20ºC for at least 2 hours. Pellet proteins by centrifugation at high speed. Residual acetone may be removed by air-drying. Then you resuspend the pellet in a lower amount of RIPA lysis buffer. By using this method, the proteins not only precipitate in a better way but also retains the structural integrity.
Best.
Perform urea-added FASP protocol (S-trap may also work). Concentrated chaotrope will disrupt the micelles and the UF membrane will concentrate the protein but also clean it by washing out the small molecules including detergent monomers. This will recover most of the precious protein, besides it is a mild way of removing bulky RIPA and matrix components...
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning