Hi
I purified 14 kDa protein with Superdex 200 pg but the A280 nm of the target protein fraction was relatively low when it was compared to the amount of injected protein mixture. The injected sample was mixture of cleaved GST and target protein. The SDS-PAGE analysis of each fractions showed the clear and well purified band of target protein. I measured the UV spectrum of target protein fraction and the peak at 280 nm was almost negligible. Then I tried BCA method for protein quantification and this method showed that the concentration of the fraction is 0.25 mg/ml. How can I think about it?
The protein contains several Tyr and Trp residues. Those aromatic acids are varied in the structure of the protein?
Did you really lose your protein or was it just diluted? And did you use the extinction coefficient specific for your 14 kDa protein? For small proteins this can differ quite a bit from the average value.
Hi there,
To estimate the yield of a purification step ou have to considered the amounts of protein after and before purification.
If separating GST from your protein of interest, yield has to be calculated from your protein of interest point of view only. In your case the protein of interest represents roughly a third of the total mass of protein injected (considering the sample contains only GST and your protein on interest) as GST is 26kDa.
Furthermore, A280 depends on the protein sequence then A280 of the sample before purification might be mainly the result of GST absorbing properties if your protein does not absorb a lot at 280nm (check the protein sequence ad process it with expasy tool for instance to get its specific epsilon at 280nm).
Thank you, Hugo van Beek and Victor David.
Yes I know the dilution of the protein. I injected 5 ml of sample and the fractions of GST and target protein are 15 ml. I always compare the integrated peak of target protein and that of GST. This time peak of target protein is 10 - 15 times lower than that of GST. I calculated the extinction coefficient of both protein and if it were to be OK the difference would be 4 times. First I thought the protein binding to the resin. But from the SDS-PAGE analysis, I could not think so. I concentrated the target protein fractions and performed BCA assay again. The concentration of the target protein was 0.7 mg/ml but the A280 nm was 0.2. I think calculated extinction coefficient is not correct. How do you think about it?
To get the extinction coefficient go to the following link and past your sequence:
http://web.expasy.org/protparam/
Why do you perform gelfiltration?
Simon
Thank you Professor Dominique Liger and Simon Arnold Mortensen.
I got the extinction coefficient of GST fused protein and GST and protein of interest by protparam to predict the A280 nm of each peaks on chromatogram before purification. And the size of the peak was much smaller than expected where the intensity of the GST peak was OK. Thus I thought the protein I want to purify bound the resin. But the SDS-PAGE analysis of each fractions especially after checking band intensities by gelanalyzer, I concluded protein was not lost. That is the reason why I doubt the accuracy of extinction coefficient from protparam. But I've never experienced the incorrect extinction coefficient from calculator thus I'm confused. Help.....
I would still like to know why you use gelfiltration.
Could you upload SDS-PAGE and the chromatogram?
Best,
Simon
Not sure what buffers you are using for gel filtration but the correct background correction should be done with the correct buffer bearing in mind the effect some buffers have on protein assays
Hi Simon Arnold Mortensen , Ian R Wilkinson
Sorry, data are confidential so I can not upload. Reason why I use gelfiltration is its very suitable to purify protein of interest especially when the molecular weight is enough smaller than GST dimer. And the buffer I used is phosphate buffer and I think this buffer is not sensitively caught by UV analysis. In addition I measured the protein UV280 using it's FT after concentration step. And I don't think buffer affected a result. I checked several papers and found the method UV 280 nm is measured in denaturing condition to avoid the effect from 2D and 3D structure. I tried and found that the extinction coefficient of protein of interest in denaturing condition is well fit to the extinction coefficient calculated on protparam (if the BCA method was correct). I tend to conclude that the the extinction coefficient in non-denaturing condition is highly affected by 3D structure.
Any suggestion and comment, Please.
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