Hi, I am working on a protein that runs in SDS-PAGE around 55kDa after Nickel purification (buffer 20 mM Tris pH8.0, 300 mM NaCl, 5% glycerol and 200 mM Imidazole), then I run through SEC in 20 mM HEPES pH8.0 and 150 mM NaCl. Overtime I see that my band starts running higher (around ~60-65 kDa) in the HEPES buffer, you see a transition after 1 day and it is totally changed after 2 days. This does not happen if the protein stays in the Tris buffer (1.5 months and it is fine). Do you know what can cause an apparent increment of the size of a protein? Does anyone has experienced something like this?
This is indeed strange.
What happens, if you bring the protein, after it has fully converted to the larger band, back from the HEPES buffer into the initial Tris buffer.
Does the mol. weight decrease again?
I have will try different conditions to point out if there is a compound in my solution that is responsible for that. Your idea is very interesting, I will try that as well!
Did you check the pH of your Tris buffer after adding the imidazole? (imidazole will increase the pH, I think) and what is the predicted MW of the protein? Proteins run in SDS/PAGE based on how much SDS the protein binds, if the protein is not fully denatured it will bind less SDS. Maybe it gets fully denatured after it is in the HEPES buffer and so binds more SDS and runs at a higher MW.
It is possible that overtime you have modification of amino acids i.e oxidation, deamination etc that may change the properties of your protein or it could be the nature of the buffer has changed the way your protein separates. Imidazole may affect your proteins properties (or other buffer components). So before heating the sample for SDS-PAGE try dialysing or buffer exchanging prior to SDS-PAGE preparation to remove imidazole and compare samples on the same gel (+/- imidazole). Also run samples in HEPES as well. This may give you an indication if it is the buffers per se or the heating of sample that is the problem.
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