protein isolation

[shweta]

hi....m tryin 2 isolate protein from fungi and profiling it by SDS PAGE electrophoresis..
but till nw i didnt get the best result....i saws 2 to 3 bands by this...
i want to know hw much fungal protein can be isolated and how...
and also wat steps i shoud prefer to get better result.in electrophoresis....
plz give me som informations...abt this.. i have never get the literature related to this by which i could take ideas.....
help me as soon as possible...

thank you...

[danfive]

See Fungal Cultures and Protein Extraction --->Evolution: Fungal Cultures and Protein Extraction

and this:
Optimization of protein extraction from Aspergillus nidulans for gel electrophoresis

Abstract: Efficient extraction of proteins from filamentous fungi is difficult both as a result of high endogenous protease activity, the existence of a hard cell wall, and the tendency of hyphae to clump. We have had difficulty using published protocols in extracting high molecular weight proteins (>150 kDa), and in extraction from germlings grown on minimal media with a poor carbon source such as glycerol. In order to address these problems, we have compared several different extraction protocols and found the protocol utilizing 9M Urea-sample buffer to be the fastest and most efficient with sample variation being kept to a minimum. ---> Protein extraction from Aspergillus

[danfive]

In case you meant protein purification:

You can use an immunopurification kit like Pierce Seize X Immunoprecipitation Kit. It allows you to fix the antibody so it doesn't elute with the captured protein (no antibody on the gel). You can also perform as many wash steps (spin cup method) as you like to get rid of highly abundant proteins that can show up on a gel as extraneous bands (for example the protocol mentions something like 5-8 washes, but one of my protocols works best with 30 washes).

[shweta]

thanks for ur suggetion ya i knw in case of fungi the mnain problem is its wall to ruptue it....ill see the site...and in case of purification the kits i cant prefer in my lab as soon i want to proceed... can i go with some other way....