Technical Support

 

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Western (immuno) blotting

Western blots of IP samples. The protein of interest has a similar size as the IgG heavy chains (~45 kDa). The secondary antibody reacts with the IgG heavy chains in the gel and masks the signal of the primary Ab used in WB. Is there a workaround?

Yes, there are two options:

  1. First of all, theoretically this should not be a problem when the antibodies (Ab) used for IP and WB are from different species (e.g. rabbit Ab for IP and mouse Ab for WB; detection with a labelled ani-mouse antibody that does not react with rabbit IgG heavy chains). However, as a matter of fact, some secondary Abs may x-react with IgG of other species, resulting in a residual reactivity with the abundant heavy chains. Therefore, you should take care to select a perfectly pre-absorbed secondary antibody.
  2. If both IP and WB are done with (the same or different) rabbit Ab(s), you may use labelled protein A instead of a anti-rabbit Ab for detection. In our hands 125I labelled protein A  proved absolutely non-reactive with Ab heavy chains separated by SDS PAGE. Protein A labelled by peroxidase should work as well. However, there are some reports that certain brands or lots may show some residual reactivity with denatured heavy chains.

Zuletzt aktualisiert am 09.08.2011 von Matthias Reinhard.

Protein MW: apparent and calculated molecular mass may differ in SDS-PAGE

The calculated MW of LPP (Lipoma-preferred partner) is 66kDa. However, in blots, it looks to be over 66kDa. Can you explain?

The apparent molecular mass of LPP (and e.g. also that of the related protein Zyxin) appear to be somewhat bigger in SDS-PAGE (~76 kDa) than calculated. This is a well-known behavior of proline-rich proteins (with stretches of 3-4 and more Pro in a row).

Due to these Pro stretches the proteins bind less SDS and consequently carry less negative charge than would be expected based on their size. Less negative charge means slower migration in SDS-PAGE. This effect often is exaggerated when there is a phosphorylation near the Pro-rich region (see corresponding FAQ).

Zuletzt aktualisiert am 27.12.2012 von Matthias Reinhard.

Why is there an apparent MW shift of certain proteins upon phosphorylation?

Proline-rich proteins (with stretches of 3-4 and more Pro in a row) bind less SDS than they should according to their size. Consequently they carry less negative charge and hence migrate slower in SDS-PAGE. This effect often is exaggerated when there is a phosphorylation near the Pro-rich region.

This effect is well know for several proteins, for example:

 

1) VASP (vasodilator-stimulated phospho protein):

  • calculated MW: 39 kDa
  • Dephospho-Ser157 (irrespective of Ser-239 phosphoryklation state): 46 kDa
  • Phospho-Ser157 (irrespective of Ser-239 phosphoryklation state): 50 kDa

2) LPP (lipoma-preferred partner):

    • The phosphorylation site causing the mobility shift is not known. However, there are several pretty good candidates:
  • pY-273 or pY-275: RGGMDYAYIPPPGLQ
  • pY-332 or pT-333: TWKREPGYTPPGAGN

Zuletzt aktualisiert am 27.12.2012 von Matthias Reinhard.