In article <30h8pi$43g at mercury.hgmp.mrc.ac.uk>, dmartin at crc.ac.uk (David
Martin x3175) wrote:
>> In article <gends.7.000D9E48 at leeds.ac.uk> gends at leeds.ac.uk (SCOTT D.) writes:
> >Absorbance provides anomalous answers, and Biorad (Bradford) assays are out as
> >the answer needs to be accurate to within 5%. Amino Acid analysis tells us
> >accurately how much protein is there, but I'd love a way of doing this in less
> >than two days!
> Umm, how about a thread on how crap most protein assays are?
> The protein I work on isn't picked up by the Bio Rad assay at all. It by
> chance contains no Histidine. are these two related.
> However, coomassie binding (on an SDS PAGE Gel), A280 (method of choice),
> and BCA all work well.
> any comments?
> any other wierd proteins out there?
Check out Kjerdahl, or some similar spelling. Very accurate, but probably
the same amount of time as aa composition (actually very similar, acid
hydrolysis, but you titrate the amount of N and figure out how much protein
based on the amount of N)
EVERY protein assay has its problems which you should make yourself aware
How about figuring out the extinction coefficient for your protein? I
that most protein assays are crap. Any protein assay is only going to give
a truly accurate estimate of your protein if you have That protein purified
use as the standard in your assay.