Great info! I'll try that. My loading buffer had 2-mercaptoethanol
instead of DTT, so I'll check the difference. I also boiled my samples
for at least 1 min before loading so I may be having a double effect.
Thanks a lot DK!
El 21/07/10 20:29, DK escribió:
In article<mailman.895.1279752425.25217.meth...@net.bio.net>, Daniel
Prieto<dpri...@fcien.edu.uy> wrote:
I have been trying to analyze some embryonic zebrafish
proteins using 1D-SDS-PAGE and western blotting, but have
experienced a very upsetting problem with some of them. I am
currently detecting bands (on the blot) as if they have
never entered the separation gel matrix. I have worked with
orthologs of this protein and the predicted sequences show
high identity so I would expect them to migrate properly.
Anyone has gone through a similar trouble? Do you think this
could be due to protein extraction and contamination with
extracellular matrix components? BTW, I'm extracting with
Triton X100 so I wouldn't expect significant DNA
contamination, nor yolk as I am manually deyolking before
homogenizing.
If it is a transmembrane protein, maybe it is aggregating when heated
with SDS. It is a known phenomenon that has never been explained
adequately. Try 200 mM DTT in the 2X loading buffer and either not
heating at all or n more than to ~ 50C.
This maybe related: I routinelt analyse soluble fraction of Trito X100
extracts on gels. There is always some material that does not enter
the gel. Definitely, the more and longer I heat the samples, the
thicker the HMW band that does not enter the gel is.
DK
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