I have purified a GST tagged protein from inclusion bodies of bacterial expression and after the purification (but not eluted), immediately, I run SDS-PAGE and I saw the band for my protein. Next day I wanted to measure the concentration of my protein using BSA standards then I see nothing in the beads. What was happen to my protein and if the protein denatured, something have to detect?
I'm not exactly sure what you mean by purified (but not eluted), I guess glutathione beads? Did your purification include refolding? However, disappearing proteins are not uncommon, especially at low concentration. Since the protein was in inclusion bodies it sounds like you are starting out with unstable protein. How did you store it overnight? My guess is the unstable protein is sticking to the sides of the plastic tube.
Hi Staker,
Yes, After thorough washing of Inclusion bodies were dissolved in a denaturing buffer (50mM Tris pH8.0, 8M urea, 20mMDTT). Then refolded with refolding buffer and then purified using GST sepharose beads. Then, with 20 microliters I run the SDS PAGE on the same day and remaining beads were stored in TBS buffer (50mM Tris pH 8, 50mM Nacl) at 4 degrees overnight.
Is it possible to collect from the walls of the tube?
If it is unstable what would be the possible way to get it properly?
Hi Sampath,
There may not be a way to get it properly. You may be able to get a trace of protein from the side of the tube, but what good would it be?
Refolding is tricky. There are different ways to refold and you should screen many stabilization buffers. It is a trial-and-error empirical process to figure out how to refold correctly and stabilize a protein from inclusion bodies.
To measure your concentration, it sounds like you will need to measure it fresh and not let it sit overnight. Sorry.
the description is a bit vague and I don't know the real situation you were having.
does your refolding solution contain GSSG:GSH? this redox system is common for refolding (to assist disulphide bond exchange). Left over GSH will dissociate your tagged protein from the column (and mess up your subsequent analysis).
if you did the purification in a tube (containing resin, thus no column was involved), your protein could actually be misfolded and when you load onto resin, you took (by chance) the aggregates. when you sampled the next day, you ended up with taking only the beads.
you said to check protein concentration using BSA standard (with Bradford? Lowry? or comparing 280 absorbance?) and saw nothing on the beads. did you measure the protein all with the bead, or you eluted first, or you run another SDS PAGE? if you measure in the presence of beads, are you sure the bead did not interfere with the measurement? if you elute it first, are you sure the protein was soluble?
Consider that your protein could get cleaved by residual proteases.
Another possibility is that the protein concentration of your next-day elution of the beads was lower than what the protein assay you used could detect.
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