Hi there,

Basically it is possible. You need two things: standard of pure protein of interest in order to calibrate the detection (I guess it's online UV monitoring) and an HPLC system and program which allows efficient separation of your protein when in cocktail with other cellular proteins.

Hi there,

Basically it is possible. You need two things: standard of pure protein of interest in order to calibrate the detection (I guess it's online UV monitoring) and an HPLC system and program which allows efficient separation of your protein when in cocktail with other cellular proteins.

Hi Sai, as Dominique noted, you can determine protein concentration by A280.

A rule of thumb is that a 1 mg/ml of a protein will give an A280 Abs of 1.

(Believe it or not, a primitive, but effective way of determining relative protein concentrations is to cut out the peaks from the paper cromatograms and weigh them).

If you can identify a specific peak as being your protein of interest and not a mixture of proteins, and if you have a standard as Dominique suggested, then you can use HPLC to quantify the protein. In a complex mixture such as a tissue sample, it may not be possible to obtain a homogeneous peak by HPLC unless the protein is a major component of the tissue. Alternatives are to do proteomics using LC-MS or 2-D electrophoresis techniques, or to set up a quantitative immunological assay such as ELISA. If the protein is an enzyme and you have a specific assay for its activity, you can use an enzyme assay to measure the amount of the protein in a tissue extract.

Before running an HPLC, I would determine the protein concentration using a spectrometer at 280 nm with the specific extinction coefficient for this particular protein. If you have the aminoacid sequence you can determine the extinction coefficient via http://web.expasy.org/protparam/ 

thank you Dominique Liger, Steingrimur Stefansson, 

Adam B Shapiro, Can Araman for all your valuable suggestions. with all your suggestion i will try for the assay. 

I have one more doubt

For standard protein- can i use commercially available peptides as standards (eg- serotonin from sigma) or should i clone the protein and purify it? 

No, you need your pure protein as standard to determine when your protein elutes from the HPLC + to make some calibration.