Unbreakable Complexes in SDS-PAGE/Western Blots?

Pow Joshi pow.joshi at gmail.com
Tue Jun 20 14:55:22 EST 2006


>I am detecting the phosporylated form of a protein by western blotting.
>
>Besides the band at the desired/expected MW (170kD), I also get a clear
>signal (which under some conditions is even stronger than the actual
>band of interest) at an apparent size of approx 250kD. And I see a
>clear time course of this band in a stimulation experiment. It is
>unlikely that this band is something like an unprocessed or partially
>processed pre/pro form. I rather suspect that there is a complex with
>some other (yet unknown) protein. I see this band with several
>antibodies against different serine phosphorylations of my protein of
>interest as well as with pY20 and total protein. I am not satisfied
>with the usual parroted explanations about precipitates, artifacts
>etc., but actually I expected that after boiling (7.5 min / 99degC)
>with Laemmli (BME) all non covalent interactions should be broken.

you could try using a "stronger" detergent, say something like sodium
deoxycholate, the one that's  present in RIPA, while you make your
"protein extract" (if you are'nt already doing it) ....

also, just a thought... would your protein have more than one
phophorylation sites? ...the mobility of the "hyper" phospho form
would of course be different ( yes, it's a large difference, in the
sizes that you've mentioned, however, I tend to believe that the
mobility is a rather empirical property depending on the charge. mass
and the compaction etc., it undergoes due to phosphorylation, which
may be resistant to SDS denaturation)......
......alternatively, do your phospho antibodies detect the non-phospho
protein, which may have a higher mobility?

pow


On 20 Jun 2006 06:24:13 -0700, Wolfgang Schechinger
<novalidaddress at nurfuerspam.de> wrote:
> Dear Experts,
>
> I am detecting the phosporylated form of a protein by western blotting.
>
> Besides the band at the desired/expected MW (170kD), I also get a clear
> signal (which under some conditions is even stronger than the actual
> band of interest) at an apparent size of approx 250kD. And I see a
> clear time course of this band in a stimulation experiment. It is
> unlikely that this band is something like an unprocessed or partially
> processed pre/pro form. I rather suspect that there is a complex with
> some other (yet unknown) protein. I see this band with several
> antibodies against different serine phosphorylations of my protein of
> interest as well as with pY20 and total protein. I am not satisfied
> with the usual parroted explanations about precipitates, artifacts
> etc., but actually I expected that after boiling (7.5 min / 99degC)
> with Laemmli (BME) all non covalent interactions should be broken.
>
> I am looking out now for suggestions on how to prove my hypothesis with
> a simple experiment (no mass spectrometry etc at this stage please) ,
> i.e. how to demonstrate that there is a complex. So, how may I disrupt
> it?
>
> All input is welcome!
>
> Wolfgang
>
> _______________________________________________
> Methods mailing list
> Methods at net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>



More information about the Methods mailing list