New form of PTM

Protein Chemistry Post-Translational Modifications
1

I have a HA-tagged protein that when expressed, show up as a 40kD protein… and there is a faint band about ~43-45kD… this faint band becomes a major band with MG132 addition to transfected cells.

So, it looks like ubiquitination…

But I cannot detect the ubiquitin (the upper band) using anti-Ubi antibody from Santa Cruz. Also tried anti-SUMO from Santa Cruz and it is not positive as well.

any ideas, advice, or experience to share?

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2

Sarah

Antibodies can vary in their action based on the epitope they have been raised against. My suggestion is to use an antibody known to detect mono-ubiquitin or pan-ubiquitin, such as:

  • P4D1 (Cell Signaling)
  • FK2 or FK1 (Enzo Life Sciences) – FK2 detects mono- and poly-ubiquitinated conjugates.
  • Apu2 or Apu3 (Millipore) – K48/K63-specific.

Also denature your sample thoroughly in SDS and DTT or TBP as Ubiquitin or SUMO modifications can be labile — insufficient denaturation can cause loss or misdetection.

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3

Sarah

Co-transfect cells with His-tagged ubiquitin or HA-Ubi, and pull down using Ni-NTA resin or anti-HA, then blot for your protein. This is a definitive test for ubiquitination.

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4

40 to 43 is a small change. It could be phosphorylated form and MG132 is simply preventing protein degradation allowing accumulation of phosphoprotein, which other wise would be a minor fraction.

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