I am attempting to calculate concentration for a protein I want to purify. I'm still unsure about what to include when calculating the extinction coefficient don't want to ask my supervisor, since he's busy with other things and I'd like to try to find the answers out on my own!
I have sequenced the construct, but as usual, there are extraneous parts of the sequence before and after the actual gene. When I did the bacteria culture and induced it with IPTG, was the entire plasmid in my BL21 cells translated into protein? If so, how am I going to be able to selectively keep just the gene of interest (or if I'm mistaken in protocols and the entire plasmid will be used, do I just use my gene's sequence in calculating extinction coefficient or the entire plasmid [which, of course, would be very large]).
Thank you all so much for your answers!
-A learning student.
Hi there,
The gene you have cloned has a coding sequence which starts with an ATG and ends up with an in frame stop codon (TAA, TGA or TAG). So expression will result in translating this coding sequence into protein sequence using the frame defined by the ATG. Processing this protein sequence will give you what you are looking for.
It should include the translated region from start to stop codon if without any tag. And if the translated protein is tagged for e.g. with hexa-histidine tag on its N or C terminal its amino acid sequence should also be considered for ProtParam analysis except if the tag is removed.
Hi,
Calculation of the molar extinction coefficient is usually performed using the following equation:
E(Prot) = Numb(Tyr)*Ext(Tyr) + Numb(Trp)*Ext(Trp) + Numb(Cystine)*Ext(Cystine)
As you can see, inclunding polyHis tag has no impact on the extinction coefficient, although it is important for calculation of the molecular weight, and thereby for determination of the molar concentration of your protein.
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