I would like to measure the concentration of a mixture of two proteins. I cannot use the absorption at 280nm because I do not know the ratio between both proteins. But one of them has a eGFP fusion domain, so I wanted to use it by measuring the absorbance at 488nm and deduce the concentration of that one protein. I found an extinction coefficient of EGFP at 488nm of 53 000 +/-4000 M-1cm-1. Is it correct to use the beer lambert law to calculate the concentration of the protein, this is to say read the absorbance A of the mixture and deduce c=A/E with E of 53000 (with the path length of the cuvette of 1cm)?
Thanks a lot for your help and comments
Yes, you can use the extinction coefficient of eGFP at 488nm. Be sure to measure at the right pH (find in literature for which pH you cited value is valid).
Remember, however, that fluorescent proteins not always come to full maturation. That would depend on the linker between your protein of interest and the eGFP, how well you protein of interest matures, in which buffers or systems your proteins are left to mature, etc etc. Sometimes, less than 20% of the FPs are matured! Be sure to also use orthogonal techniques to check this!
Thanks for your answer Benjamin, and for the warning about pH, it is true that extinction coefficients can vary a lot with pH.
Also, I am not so experienced with working with GFP, it is my first construct. Actually it is added after an intrinsically disordered small domain of 85 amino acid containing a streptag at the C terminal, and a short linker GLEGG.
When you say not all GFP are mature, it mean that I could capture non mature GFP with my streptag and measure false concentrations?
I will look in literature how to perform these orthogonal techniques then!
Thanks again
Dear Anabel-Lise,
Indeed, you'll capture immature fluorescent protein, but not detect them in the spectrophotometer (at least not at ~500nm).
Depending on the fusion construct, you could get anywhere between no folding/maturation at all to full folding (eg. Pédelacq, Nat Biotech 2006).
For a "quick and dirty" validation I'd do an SDS-PAGE and include some standards. Stain with eg. Coomassie and assess the validity of your data. You could even use some analysis software to make a calibration curve and judge your concentration somewhat more precise.
B
Dear Benjamin
Thanks for your tips; actually I wa thinking that if I manage to separate the protein complex I have and isolate the protein-GFP, I should be able to compare both concentrations derived from 280nm and 488nm, and derive if I have a lot of non mature protein or not! I hope it could happen! Thank you very much!
Anabel-Lise
I think this link is very useful,
http://www.instanano.com/characterization/theoretical/concentration-calculation-from-uv-vis-absorbance/
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