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: CCP4BB@JISCMAIL.AC.UK >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/signatur >e-home.htm/[EMAIL PROTECTED]/2206641_2199021/2201650/1?PARTNER=3&OAS_QUERY= >null> Rediff Shopping > > >
