Hi Folk,
I want to share my experience and unanswered issues I have with a protein. It is a protein known to bind a 2Fe-2S cluster and/or Iron III. The protein is eukaryotic from mitochondria, here expressed in Ecoli with a fusion partner. The protein aggregates/precipitates too when purified and concentrated, and some Nter specific cleavage appeared over time -but not observed when purified and kept as a fusion protein (should be contaminant proteases but still wondering if it can be an iron mediated thing??)
But my main problem here is that the UV absorption spectra change over time. Here you have an example over 4 days in the attached file. After few hours the sample has precipitated heavily. The next 2 measurements are with supernatant of that sample. So my question is what can possibly cause my absorption maxima to decrease/increase as observed????
Thanks,
Guillaume
I think the answer is in your question itself. In general, amount of protein is directly proportional to the absorption. You mentioned that, the protein is precipitated which influenced the concentration and hence the absorption. And also, the lower the protein concentration in a solution, the additional factors (salts and other components of the solution) may influence the absorption of the solution as a whole. Goodluck
Thanks for the comment Ramachandramouli. I see, I'm not that used to work with difficult proteins... It make sense that precipitation induces a decrease in absorption, but what kind of factor/mechanism can make a direct increase of the absorption at a particular wavelength?
I mean, there is a blank made with the protein buffer...
The main factor is the spectrometer itself. The lasers and the photomultipliers in the instrument are not always same. They behave bit different every time the machine is turned on. And so, the protein estimations made by these machines is only relative measurement to the blank or control absorption values. That is the main reason, may factors (such as salts, DNA contamination and others in the sample buffer) influences the measurements which are not present in the blank buffers. I hope I could give a proper explanation. Good luck
Hi,
I figured out that the problem is not coming from the spectrophotometer or contaminants with sample buffer.
I can see a late coming peak on gel filtration for the protein sample with an increase of absorption over time at 260 as well as 280 nm.
I also did set-up some controls with BSA and I observed the same phenomena after few days for BSA WITH beta-mercaptoethanol. Both are needed somehow as the blank is with betaME too....
- Badges
- Science topic
- Similar topics
- Microbiology
- Assays