Dear Madeleine

the buffer that you use for extraction depends from the purification approach thay you would like to use and the properties of your protein eg isoelettric point.

generally for ionic exchange you need to extract the protein with a buffer with no or low salt and a ph different at least 2ph units from your protein pI to charge the protein and make it able to bind the coloumn. eg protein with pI=8 you can use phospate ph6.5 or mes ph 6 and perform cationic exchange.

Other purification approaches as imac allow the presence of Salt and therefore you can add it to the buffer.

freeze-thawing may work only in very small scale. i dont know which is your culture scale and amount of protein that you need to purify but take in consideration that to acheive good extraction Stronger approaches as sonication may be necessary.

if you do not have access to a sonicator a possible interesting alternative lysis agent is cell lytyc express ( sigma cod c1990) which contain chaps and lysozime, it is compatible with imac and i think also with ionic exchange. you need

just to resuspend your pellet, incubate 30' at room temperature under sharing, centrifuge and recover soluble proteins in the surnatant.

i purified with it several enzymes that ( which where not unfolded from the chaps). the only drawback is thr cost. it is expensive in scale up because to acheive good lisis you need at least 20ml of it for each g of wet biomass.

good luck

Manuele

Sonication along with lysozyme treatment is very useful, often used in addition to freeze/thawing, assuming lysozyme is not counfounding for your specific protein. You would do well to isolate the insoluble fraction if theres a chance your protein of interest (POI) could form inclusion bodies (IBs). Recognize that unlysed cells co-sediment with IBs so VERY through lysis is required to not get a false positive for insolubility. I might include a nice neutral 20-50mM HEPES buffer, 200mM NaCl initially. I'd start mild on the lysis buffer, only escalate if needed as chaotropes/detergents etc. can really mess with downstream assays. Good luck!

I would initially start with a simple buffer like Tris Buffered Saline (TBS) with 50mM Tris, pH 7 4, 150mM NaCl. If necessary and a non-ionic deterfent like Triton x100 or Nonidet P40 at 1%.

Unless you are lysing a pLys S strain, freeze-thaw alone will not be too efficient unless you add lysozyme or you sonicate or French Press to help it along.

During lysis and assays for proteolytic activity, you will need to keep in mind not only the stability of your protease of interest with respect to buffer conditions. You will want to know about proteolytic degradation by other proteases and even by itself, i.e. one active molecule of your protease of interest performing proteolytic digestion on another such molecule. If you have the sequence, you can perform a BLAST search to determine what class of proteolytic enzyme you are working with. Knowing this will give you clues on how to design your first attempt at lysis. Add inhibitor or inactivator of other classes of proteases. For instance, if the sequence suggests that it is a serine protease, you can help prevent metalloproteases from degrading your protease of interest by adding EDTA. You can prevent cysteine proteases from degrading your protease of interest with iodoacetate and E64. If your sequence suggests that you have an aspartic protease, which typically have acidic pH optima, work at neutral pH. You might need to add DTT to your assays if the sequence suggests that you have a cysteine protease. The BLAST search might even lead you to a finer level, e.g. leading you to published articles that will serve as a better guide for that first attempt. After you have a lysis method that yields active protease, you can refine your method for further studies.