Hello everyone,
I am working in protein extraction of acetic acid bacteria and sometimes I have problems in the solubilization step. After cell lysing, washing protein extracts (DTT-acetone) and vacuum dry, I try to solubilizate the dry protein extracts using a solubilization buffer composed of 8 M Urea, 20 mM DTT, and 20% CHAPS prepared in distilled sterile water. After applying this buffer, I proceed with numerous vortexing and shaking cycles, and normally, a lot of foam is produced but small protein solutes remain in the buffer without being fully solubilized. This problem can remain this way for hours, so I suspect that my solubilization buffer may need some modification or addition of components.
Can any expert propose a solution to this problem?
Thank you
Juan J.
"propose a solution" to insolubility ? ; ) Jokes aside, there is no perfect solubilization method across all cell types or bacteria, just general conditions which are fine tuned for your specific cells. Acetone precipitation if quite harsh; there are proteins which will be impossible to make soluble again IME. In terms of "most aggressive solubilization buffer" GuSCN/TCEP/SDS works quite well for some downstream applications, but kills others, like immunoassays (where RIPA usually works better). What exactly is the downstream analysis you are hoping to do? What works for a Lowry assay is often useless for MS etc.
Try to use B-PER™ Bacterial Protein Extraction Reagent. This is very convenient to solubilize most of the proteins from bacterial origins and it is also compatible with further analysis in terms of many downstream approaches. Otherwise, you may try to increase the detergent percentage or ionic (Such as SDS/LDS) ones must be preferred to improve solubility. There is a trade-off here unfortunately when the solubility is increased by ionic detergents you would lose the high order structure therefore its activity too...
I would just like to point out that your "buffer" is not really a buffer because it contains no buffering agent, or at least none that is typically thought of as a buffering agent (the pKa of dithiothreitol is around 8, apparently). It might help to add a pH buffer at around 7, such as HEPES-NaOH, 50 mM to mimic a typical intracellular pH.
However, I think a bigger benefit would come from avoiding acetone precipitation/lyophilization. You can store the cell pellet at -80oC if necessary, rather than making an acetone powder.
Edward Michelini Adam B Shapiro After cell lysis (glass beads and extraction buffer), I make a TCA-Acetone-DTT (0.07%) precipitation at -20ºC overninght (12-15 h), then I discard the supernatant and continue making a double washing with only Acetone-DTT of protein extracts and follow the above procedure. Perhaps the problem is the hardness of the precipitation which insolubilizes many proteins... and not the solubilization buffer...
Maybe I can reduce the volume or time of precipitation (TCA-Acetone-DTT)...or can I directly modify/delete this step?
Edward Michelini My downstream analysis is LC/MS-MS by shotgun for identification of proteins and making metaproteomics.
Amphipol A8-35 would be helpful in the resolubilisation step after aceton precipitation of the proteins. Amphipol is compatible with trypsin in the step of digestion and would be carefully use in down-stream analysis by LC-MS. After digestion you can precipitate amphipol using 5% formic acid solution prior to LC-MS
- Badges
- Science method