BME / DTT Immunoblot

Wolfgang Schechinger wolfsc at ibms.sinica.edu.tw
Sun Apr 29 11:15:29 EST 2001


OK, here we go: 

I assumed that after transferring the proeins to the membrane, 
some reducing agent (BME or DTT) will be present on the menbrane 
(or bound to the proteins) and even after several washing steps, 
some would prsent, normally not affecting yor signal in ECL. 
however, when signal is very low, BME still present might render 
your antibodies ineffective.
DTT has two SH groups present. So, when when there are oxidizing 
conditions, it is very likely that ring formation will take 
place since there *always* a second SH group is present, even 
at very high dilution. In contrast, when you 
have BME, then it is not so  likely that a second SH group is 
present near a given BME molecule and thus such a BME molecule 
is more likely to form an -SS- bond with a Cysteine SH group and 
thus maybe might compromise the antibody.

The thought of impurities as the cause is interesting to me. I 
never thought of that possibility. Do you have informations on 
the nature of these bad guys? There might be oxidises products 
("dimers", and sulfoxides, but those should not harm much). what 
else could be present?

Wolfgang

> 
> > DTT may be favored aginst BME in your case since the two SH 
> > groups present will more likely react with themselves when they 
> > become  oxidized at a high dilution (as the conditions in 
> > western blot will be). BME should be more likely to react with 
> > other molecules (of BME, of antibody or of protein, whatever is 
> > present). 
> 
> I am not completely understanding of this explinantion.  In my case, BME 
> is a poor reducing agent and for reasons unknown to me (but I 
> speculate), when I prepare my samples in BME, I loose signal,  and I 
> mean a lot of signal (at least factor of 10).  A freind of mine who is  
> a protein chemist told me something about BME that may be relevant.  
> Most of the preperations of BME that are purchased are not very clean 
> and may contain other chemicals that will covalently attach to your 
> protein. 
> I suspect, The rate of disassociation of BME with a protein is more 
> likely to occur than the disassociation of DTT from a protein.  I know 
> that DTT is considered to be a more potent reducing agent than BME and 
> it works at room temperature.
> 
> regards,
> 
> 
> 
[tiss mezzage wahs broduceRd using TYPO GENERATOR zoffwer]
-----
Dr. Wolfgang Schechinger
Lab N233 (c/o Dr. Steve Roffler)
Institute of Biomedical Sciences
Academia Sinica
128 Yen-Chio Yuan Rd. Sec.2
Taipei 115
Taiwan R.O.C.
Tel +886-2-2789-9152
Fax +886-2-2782-9142
Mobile +886-925-136893
wolfsc at REMOVETHESEANTISPAMCAPITALSibms.sinica.edu.tw
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