I am unable to find any proper bands in the 15% gel lower to 10KD. My protein of interest is of 5-8KD. Is there any resolving problem in my gel?
Try the tricine gel system. "Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.
Schägger H, von Jagow G. Anal Biochem. 1987 166(2):368-79.
it's easy to run such small proteins off the bottom of the gel, especially when trying to get good resolution so be careful not to run it too long. Also, remember that small proteins stain less well, so the band might be fainter than expected.
You could get anti-his antibodies and do a western.
For tricine sds Page get the full protocol here... i think its better then the original publication in terms of experimental ease.. http://www.nature.com/nprot/journal/v1/n1/full/nprot.2006.4.html
Hi Poulami, as others suggested, tricine gels will probably be your best choice to retain peptides smaller than 10kDa on SDS-PAGE.
But how are you detecting your His-tagged peptide? Are you doing westerns and using anti-His tag Abs?
i would suggest to try the tricine gel system but then a Western blot with antibodies specific for the His-tag should be used.
Try Tricin gels coupled with western or silver staining. If you do not find a good resolution, i would suggest that you shall buy pre-catsed Tricin-gradient gel (assuming that you people do not have apparatus to cast the gradient gels).
Check BIO-RAD. They also sell quality pre-casted gels.
@Steingrimur and all,
I am trying to express my protein in BL21 by using 1mM IPTG. So, before western I an trying to figure out that my particular protein is being induced or not.
Hi Poulami,
are you also loading the cell pellet on the SDS-PAGE? maybe this concentration of IPTG is too high and this can create stress in the cell and the protein will be produced in high amount and inclusion bodies will be formed and you cannot see any protein in the soluble fraction.
Then I would anyway try to load both of the fractions (unsoluble and soluble) on Tris/Tricine gel instead of the normal SDS-PAGE gel because of high resolution of small peptides (like yours) and anyway also Western Blot can confirm the presence of your protein of interest using antibodies against the His-Tag (you can find from Sigma different ones).
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