Protein Concentration Estimation

berry at biochem.unp.ac.za berry at biochem.unp.ac.za
Mon Jul 25 04:28:59 EST 1994


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:
> ...d
>
>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.


>takes
>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
>of.
>How about figuring out the extinction coefficient for your protein?  I
>disagree
>that most protein assays are crap.  Any protein assay is only going to give
>you
>a truly accurate estimate of your protein if you have That protein purified
>to
>use as the standard in your assay.
>
>Ginnie


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).

Ron Berry
Biochemistry Dept
University of Natal
Pietermaritzburg
South Africa
berry at biochem.unp.ac.za



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