mercaptoethanol ghost band in Western

Richard Kerr kerrr at CRYPTIC.RCH.UNIMELB.EDU.AU
Mon Sep 7 19:01:25 EST 1998


At 07:23 7/09/98 -0700, you 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
>------------------------------------------
>
>Possible solution:
>
>We sometimes have detected the same problem with reduced 
>protein samples, although we work on bacteria. It has to do something 
>with the quality of the mercaptoethanol ("chemical aging"). To 
>avoid this problem, we put mercaptoethanol at  -20 C for an hour or 
>so, and then try to recover the less viscous fraction, leaving behind 
>the floculated portion that will become evident if mercaptoethanol is 
>indeed the problem (too old, usually). Then we use it as normally 
>in the sample buffer for SDS PAGE or any other. 
>Hope this will help,
>
>ORLANDO LUIS PARDO LAZO
>DIV VACUNAS, CIGB,
>C. HABANA, CUBA. 


ANOTHER POSSIBLE SOLUTION
if it is indeed the age of your beta-mercapto, then try another reducing
agent.  Dithiothreitol (DTT) or dithioerythrit (??) DTE also reduce proteins
nicely without the ghost band problem or the stink of betamercapto.  ONE of
these reagents is also called Cleland's reagent...but I don't remember which
one.....
hope this helps.
Richard Kerr.
The Murdoch Institute,
R.C.H. Flemington Rd, Parkville, 3052,
AUSTRALIA.
kerrr at cryptic.rch.unimelb.edu.au
Phone (61) 3 9345 5045.
FAX   (61) 3 9348 1391.
'The most interesting things about vertebrates occur in the neural crest.'
	Peter Thorogood.




More information about the Methods mailing list