I have a resolubilisation problem with serum samples. 250µl serum was
500µl lysis buffer (8M Urea, 100mM TrisBase, 2% w/v SDS) and centrifuged at 16.000 x g for
centrifuged at 16,000 x g for 5min. The supernatant was pipetted into a new eppi
and 100µl TCEP (10mM in H2O) was added. After vortexing and
centrifugation (5min at 16.000 x g) the supernatant was lyophilised in a new eppi.
lyophilised.
Yesterday we first added 500µl of Milli-Q water to the pellet to dissolve.
However, a firm gelatinous consistency was formed. Then we added
500µl of Milli-Q water again, but the sample remained gelatinous.
Vortexing, shaking at 33°C in a thermo shaker, freezing and thawing,
Ultrasonic bath and lance didn't help.
Regards
There is a very large amount of protein in serum (mostly albumin and immunoglobulin).The protein was completely denatured by the lysis buffer because of the urea, SDS and basic pH. It's no wonder that it formed a gel. It's basically the same as cooking it: the proteins unfold and coagulate.
What was the purpose of the lysis buffer and TCEP? Why not just freeze-dry the serum as is? (Also, why add the TCEP in a second step instead of at the start?)
Assuming there is a good reason for your procedure, I would suggest that diluting the serum at least 10-fold before adding the lysis buffer might help keep the denatured proteins from coagulating because there would be sufficient urea and SDS to keep them in solution.
Hi Shahid Aziz . Can you be more specific in your question? Plus, the copy/paste of your protocol is difficult to follow.
I have to agree with Adam B Shapiro , it's hard to understand the purpose of what you have done.
Serum is the cell free component of blood, as such lysis buffer is unnecessary, you have nothing to lyse.
It would be good to understand the goal of your process, what are you trying to achieve?
With the detergent and urea in your lysis buffer, you have unfolded the proteins in your sample. The tris(2-carboxyethyl)phosphine has reduced -S-S- bonds, further unfolding the protein.
Serum is about 70 mg/mL protein, you diluted this 3-fold with sample buffer, and then slightly with the TCEP. Thus, the final concentration is about 20 mg/mL, which is very high.
Imaging you mix many long threads of yarn and shake them about good. This is a reasonable model for the protein in your sample. How easy will it be to pry them apart?
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