I am purifying 8 Kda Protein tagged with GST. My 8 Kda protein don't have any tryptophane. So for calculation the concentration of my fusion protein I need extinction coefficient of GST. Please tell me the extinction coefficient of GST in 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.0 Buffer.
1. http://web.expasy.org/cgi-bin/protparam/protparam
2. http://www.biomol.net/en/tools/proteinextinction.htm
I am not much sure but what I feel like you are using water as a solvent in your buffer, so whatever value of GST coefficient will be in water, nearby value you can assume because I don't think somebody have optimized coefficient value in different buffer system with different protein. so just have a look and good luck.
By doing a simple Google search you can get that info.
From this booklet,
https://www.google.com.mx/url?sa=t&source=web&rct=j&ei=fLXKU_jHCMai8AGc3ICQAg&url=https://www.gelifesciences.com/gehcls_images/GELS/Related%2520Content/Files/1314787424814/litdoc28960573AA_20110831144748.pdf&cd=2&ved=0CB0QFjAB&usg=AFQjCNEzLd7CGFYAezCN6qGCXy-qaN1plw&sig2=1SJk8LD145q6mXIsXuxVfQ
Hi; Here is a method in a paper form Pace at al. for calculating the extinction coefficient in water. I include the abstract from that paper for your information.
Protein Science (1995), 4:2411-2423. How to measure and predict the molar absorption coefficient of a protein, by C. Nick Pace, Felix Vajdos, Lanette Fee, Gerald Grimsley, and Theronica Gray
Abstract: The molar absorption coefficient, E, of a protein is usually based on concentrations measured by dry weight, nitrogen,or amino acid analysis. The studies reported here suggest that the Edelhoch method is the best method for measuring E for a protein. (This method is described by Gill and von Hippel [1989, Anal Biochem 182:319- 3261 and is based on data from Edelhoch [1967, Biochemistry 6:1948-19541.) The absorbance of a protein at 280 nm depends on the content of Trp, Tyr, and cystine (disulfide bonds). The average E values for these chromophores in a sample of 18 well-characterized proteins have been estimated, and the E values in water, propanol, 6 M guanidine hydrochloride (GdnHCI), and 8 M urea have been measured. For Trp, the average E values for the proteins are less than the E values measured in any of the solvents. For Tyr, the average E values for the proteins are intermediate between those measured in 6 M GdnHCl and those measured in propanol. Based on a sample of 116 measured t values for 80 proteins, the E at 280 nm of a folded protein in water, E(280), can best be predicted with this equation:
E 280(M-1/ cm-1) = (#Trp)(5,500) + (#Tyr)(1,490) + (#cystine)(125).
These E(280) values are quite reliable for proteins containing Trp residues, and less reliable for proteins that do not. However, the Edelhoch method is convenient and accurate, and the best approach is to measure rather than predict E.
Bjarni's answer is excellent. I know the Pace paper and have used it.
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