Hey,
There are some calculation you can predict the amount of epsilon (molar extinction coefficient) by amino acid content. You may also prepare a solution of the protein of interest with known concentration and by applying this into beer-lambert's formula (A=eCl) obtain the epsilon. So, what's the difference and which one should I use?
Thank you in advance.
I have determined insulin concentration by HPLC-photometric detection (please see file; Insulin RP-HPLC). Lambert-Beer law says that direct photometric determination without purification is not good. Therefore, ELISA method also gives false determination values.
Thank you Dr. Kou Hayakawa
Due to what you said, I gather that the impurities cause the difference between two amounts of epsilon.
In a case of pure protein, which method should be applied and why?
Molecules DNA and protein/peptides can aggregate to dimer or multimer. Then, molar extinction coefficient may change in spite of pure condition.
RP-HPLC-photometric method is reliable to measure monomer molecules.
Then, I would like to recommend the use of HPLC method again.
Apply Edelhoch method, and you should be able to apply spectroscopy A280 nm to measure protein in solution.
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