Non-specific immunodetection

Frank O. Fackelmayer Frank at Fackelmayer.de
Mon Nov 19 13:01:11 EST 2001



Petr Brauner wrote:
> 
> After years of doing immunoblotting, I used an antibody of one of my
> friends. Although I used recommended titers of primary and secondary
> antibody and the same washing conditions as well, I got absolutely
> non-specific image. What is even worse, this non-specific image appeared
> even in the negative control (i.e. WB without primary antibody).


which tells you that the secondary antibody is to blame. I would try
diluting it more (at least a factor of 10 more, possibly even 50 to
100), and/or adding milk powder to the secondary antibody dilution at a
concentration of 0.5%.


> When we
> compared our protocols, the only significant difference is that I do not
> equilibrate SDS gels prior to mounting them to the blotting (semi-dry). I
> only wash them very briefly. My friend allows them to equilibrate for 30
> min. 

which is usually not a good idea because the proteins are not fixed in
the gel and at least the smaller ones diffuse out rapidly.

> Does anybody know whether this could possibly affect the
> immunodecoration? 

it should not

>Another question - is equilibration of the gel necessary
> to perform blotting or not? If so, how long?

we never do it, and not even rinse the gel before assembling the blot sandwich.


> If I omit the equilibration,
> what it may cause?

stronger signal, as your protein will not have diffused out of the
gel... :)




Frank




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