mercaptoethanol induced ghost band in western-blotting with rabbit antisera

Dr. Peter Gegenheimer PGegen at UKans.nolospamare.edu
Fri Sep 11 19:34:30 EST 1998


On Mon, 7 Sep 1998 09:44:32, Ulrike B÷er <uboeer at gwdg.de> wrote:

> Question
>
> Our group is working about G-protein coupled prostaglandin rceptors and
> our aim is to detect receptor proteins by western-blotting using
> polyclonal rabbit antisera against the receptors. Unfortunatly, if we
> reduce our protein samples to denaturate the receptors we see a very
> prominant band with a molecular weight around 67 kd after treatment of
> our blots with rabbit antisera wich is not vissible if we do not reduce
> our samples.
> I would like to ask if someone have also observed this phenomenon and
> have overcome this problem?
> Thank you very much.
> --
> Dr. Frank Neuschaefer-Rube
> Dept. of Biochemistry
> Georg-August-University, Goettingen, Germany
> Email: fneusch at gwdg.de
> Tel: xx49 551 39 5950 / 5978 Fax: /5960

As another poster mentioned, one source of artifactual bands at 50-70 KDa is
keratin. I know a number of posters to this group have singled out
mercaptoethanol itself as the culprit. However, after reading some of the other
posts in response to yours, I am tempted to speculate: since keratin fibers are
crosslinked via disulfide bonds, mercaptoethanol might release soluble keratin
from dead skin cells, hair, or fingernails.

We have seen these "artifactual" bands both by silver and by Coomassie staining
(not with an antibody). In our case, it was a band of ca. 55 KDa whose sequence
showed that about 1/3 of it was human keratin. The sequencing facility (Keck
Center at Yale U, New Haven USA) said that they had seen several samples of
about this size which were human keratin.

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