Hi, Thank you for all replies.
Sincerely Debajyoti On Mon, 25 Aug 2008 Artem Evdokimov wrote : >If your protein precipitates at low ionic strength you may want to establish >what concentration of salt does it tolerate (by slowly diluting a fraction >of your pool with water just until you see precipitation) then try to adjust >your cation exchange conditions so that you load with enough salt to keep >the protein happy, then elute with higher salt concentration. You may want >to adjust the pH of your buffer to be lower - usually this helps keep things >in solution with basic proteins such as yours. In general you don't *have* >to dialyze prior to ion exchange - dilution works just as well. > > > >Imidazole and histidine are pretty much the same in terms of protein >behavior, at least in my experience. Both of them can cause 'apparent' >protein degradation - if you take a sample of your protein with histidine or >imidazole still in it, then boil it with the gel loading buffer you can get >fake proteolysis (i.e. only in the boiled sample). It does not happen with >every protein, but it does happen (some weird chemistry involving the >imidazole ring at high temp. no doubt). > > > >Artem > > _____ > > From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of >Debajyoti Dutta >Sent: Sunday, August 24, 2008 3:21 AM >To: [email protected] >Subject: Re: [ccp4bb] protein degradation > > > > >Hi again, > >Thank you for all you have replied. I suspect the sonnication for such a bad >result. I am just wondering if I can use Histidine instead of Imidazole and >then buffer exchange to go for Cation exchanger. >Does Imidazole had any bad effect in protein. I have experienced that the >protein cannot sustain low salt and get ppt during dialysis. > >Sincerely >Debajyoti Dutta > > >On Sat, 23 Aug 2008 Artem Evdokimov wrote : > >Hi, > > > > > > > >If you are plagued by 'generic' proteolysis, it is not likely that changing > >buffers from TRIS to phosphate will help reduce the breakdown. You may want > >to ask yourself several key questions regarding the breakdown: > > > > > > > >1. is it proteolysis or abortive translation? > >2. is it happening during expression or post-lysis? > >3. is it exoproteolysis or endo- (or both)? Which terminus is more > >affected? > > > > > > > >If you have abortive translation or alternate transcription/translation > >starts, you should examine the DNA/RNA sequence of your gene closely - > >sometimes you can tell (e.g. beginning of the gene is peppered with > >methionines and S-D sequences) > > > > > > > >If you have proteolysis during expression, it sometimes can be alleviated >or > >even eliminated by changing expression conditions (temperature and richness > >of media seem to be key - this summer we had exactly this kind of a > >situation where expression at 37C for 4 hours gave more total protein than > >expression at 20C overnight, however the 37C protein was significantly > >'busted up' whereas the 20C was essentially intact). > > > > > > > >If you have proteolysis during cell lysis you may be able to reduce its > >extent by means of at least the following (not a complete list by any > >means!): > > > > > > > >a) lyse and process the cells on ice or even in liquid nitrogen (see > >some previous posts regarding mortar-and-pestle LN2 lysis). Avoid >sonication > >or other forms of mechanical lysis in-liquid as they all generate heat > >(detergent lysis or French press may be safe alternatives) > > > >b) make an effort to lyse the cells and complete primary extraction as > >fast as possible (for example, in an extreme case you can forego the lysate > >clarification step - high-flow IMAC resins can tolerate complete crude > >lysate in batch mode or sometimes even on a column). > > > >c) use protease inhibitors (can be somewhat expensive) > > > > > > > >If you can figure out which end of the protein is affected (or perhaps its > >lysis of an interdomain linker, exposed loop, etc.) you may be able to > >re-design the construct to avoid this. Presumably the fact that your >protein > >can be extracted via the His6 tag means that the end with the tag is intact > >(or at least enough of it is intact to make purification possible). > >Therefore it may be interesting to switch the tag to the other end - you >may > >get lucky. > > > > > > > >If you cannot afford the (expensive) commercial protease inhibitor > >cocktails, you may be able to get away with using the 'old basics' such as > >benzamidine, PMSF (AEBSF), and EDTA/EGTA or phenantroline. Yes, you won't >be > >able to use IMAC together with EDTA - but you can always do cation exchange > >first (protein with pI of 10!!!) in EDTA, then polish it up with IMAC etc. > >If your protein is really pI 10, it should bind to CM-sepharose in buffer > >with pH as high as 8, which is essentially a guarrantee that your protein > >will be almost alone in the extracted material - you wil likely see a > >ribosomal p10 protein that's about pI 11 - and that's it. Remember that if > >you go this way, you should avoid using lysozyme for cell lysis since HEWL > >is mighty basic itself. > > > > > > > >Good luck, > > > > > >Artem > > > > > > > > _____ > > > > From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of > >Debajyoti Dutta > >Sent: Friday, August 22, 2008 6:16 PM > >To: [email protected] > >Subject: [ccp4bb] protein degradation > > > > > > > > > >Hi, > > > >This is going to be an off topic question concerning this community. I have > >a protein 6XHis tagged. When retrieved from the Ni-NTA column with >imidazole > >found to be degraded, appears like a deep band with other bands (touching > >each other below the main band) in SDS PAGE. The protein is a DNA binding > >(pI~ 10) and Tris-Hcl buffer is used with 10% glycerol and 300mM NaCl. for > >purification. Does Phosphate buffer do any help in stopping the >degradation. > > > >All suggestions are welcome. Thank you for your reply in advance. > > > >Sincerely > >Debajyoti Dutta > > > > > > > > > > > ><http://adworks.rediff.com/cgi-bin/AdWorks/click.cgi/www.rediff.com/signatu >r > >e-home.htm/[EMAIL PROTECTED]/2206641_2199021/2201650/1?PARTNER=3&OAS_QUERY >= > >null> Rediff Shopping > > > > > > > > > > > ><http://adworks.rediff.com/cgi-bin/AdWorks/click.cgi/www.rediff.com/signatur >e-home.htm/[EMAIL PROTECTED]/2206641_2199021/2201650/1?PARTNER=3&OAS_QUERY= >null> Rediff Shopping > > >
