Protein characterization - what kinda stuff do u do? (Aug/15/2006 )
I wanna know what ppl in labs do in terms of protein characterization, what techniques are followed?
Also, what forms of forced degradation protein products like oxidation etc are targetted. and what methods are employed for these, i.e. the generation and detection of these?
Depends on the protein
i have purified a novel enzyme and did the following things to help characterise it:
* effect of temperature and pH on enzyme activity
* amino acid content by gas chromatography
* effect of metal ions on activity
* inhibition characteristics
* enzyme kinetics
* substrate specificity by proteolytic digestion for bonds formed by the enzyme
* also did sequenced a few fragments from a tryptic digest and got them sequenced to enable m to design some primers to get gene by PCR
Just a few ideas, if you think about what your specifically your protein does then you can decide on the best tests though some are a must!
To degrade your protein i would use some chemicals that cleave at specific sites (CNBr, V8 protease trypsin, chymotrypsin etc etc etc), dependingon the relative amounts of these you ay be able to separate them by SDS-PAGE if not then perhaps by HPLC or Mass spec if you have access to these
hope this helps