More thoughts: Extracting soluble and insoluble protein fractions
Most popular method is bead-beating. Advantages: fast, cheap, widely used. Disadvantages: messy, incomplete lysis, evidence of protein denaturation?
Zymolyase works cleanly and with high efficiency. However, it requires a long incubation period (30-45min) during which we see evidence of protein degradation. Unclear if this is a stress response or something else.
Chemical lysis is an attractive route for extracting yeast proteins, because enzymatic treatments leave extraneous proteins in the sample and mechanical lysis, e.g. with beads, is a bit of a pain. While several companies sell chemical lysis reagents, such as Novagen's YeastBuster and Pierce's Y-Per, the formulations are proprietary and it's not clear what the tradeoffs might be.
The major active ingredient in Y-Per lysis reagent is apparently 10 mg/mL SB3-14 (myristyl sulfobetaine), also known as 3-(N-tetradecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate or SB14, available from Sigma as SB3-14. Y-Per is a protein-free reagent and SB3-14 is the only detergent .
For assessing soluble versus insoluble fractions of proteins, chemical lysis of membranes may not be ideal. The problem is that if detergents liberate proteins from the membrane efficiently, then we can expect them to solubilize some insoluble proteins, and reduce signal. The appeal of mechanical lysis is that it is not expected to alter the solubility of proteins.
Idea: Mechanically disrupt, spin, and extract supernatant. Spin down to clarify. Draw off supernatant as soluble fraction and save pellet. Wash beads with 100 μL solubilization buffer, vortex, and draw off as much material as possible (liquid + pellet). Add to pellet remaining after soluble fraction removal. Vortex to resuspend and incubate 5 min at room temperature. Spin down to clarify. Draw off supernatant as insoluble fraction.
SUME Buffer (1% SDS, 8M Urea, 10mM MOPS, pH 6.8, 10mM EDTA) To make 100ml:
* 1.0ml 1M MOPS, pH6.8 stock solution * 1.0g SDS, or 10ml 10% SDS stock solution * 48.05g Urea * 2.0ml 0.5M EDTA stock solution
This is used for lysing yeast at pH6.8. For pH8 lyses, use the variation of SUME with Tris buffer known as SUTE.
from Yeast Protocols
- Zanna L and Haeuw JF. . pmid:16978932.