In article <Dress-220794113146 at chuck.biosci.arizona.edu> Dress at biosci.arizona.edu (Virginia Dress) writes:
>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:
>>Check out Kjerdahl, or some similar spelling. Very accurate, but probably
I really don't think the kjeldahl method will be sensitive enough. ALso you need specialised equipment for doing the digestion. In addition it is a tedious method unless you have a facility already set up, and someone else is going to run them for you. To do one or two a day is not worth setting up.
>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.
I agree with Ginnie - EVERY protein assay has its drawbacks.
FYI, I posted David a document describing a student prac that is designed for EXACTLY this problem - protein determination in the presence of DNA/RNA.
It relies on the fact that the UV spectrum of nucleic acids goes through a minimum at around 230nm, whilst between 260 and 200, proteins show a great increase in UV absorbance. By reading at DNA/RNA isoabsorbance points on both sides of the minimum, any increase in absorbance is due to protein.
Being a spectroscopic method, it is quick, easy and reproducible, and with micro-cuvettes doesn't need much sample (which is also recoverable, unlike aaa, kjeldahl, etc). Most modern instruments let you read at a couple wavelengths, and may even do the subtraction for you ...
The reference is:
Groves, WE, Davis, FC and Sells, BH (1968) Anal. Biochem., _22_, 195-210.
If anyone wants a copy of our protocol, I will fax or E-mail with pleasure (MS Word for Windows format, but MS Word for Mac or WordPerfect for DOS can be arranged).
University of Natal
berry at biochem.unp.ac.za