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I ran across this paper recently.

http://www.ncbi.nlm.nih.gov/pubmed/21151122

I am confused on their use of Western blotting. Their unmodified protein (no PTM group) has a band and their modified protein (succinylation of Lys) has no band, yet they use this as evidence of successful identification of the PTM. Can anyone see what they are doing here?

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They are doing a peptide competition assay (see abcam protocol).

The antibody is incubated with a purified sample of the antigen and used in a normal Western-Blot of cell lysates. Any cross-reactivities of the antibody would still be visible as only the interaction with the epitope of interest is blocked. So all bands that disappear after such treatment are indicators for the substance that was used for blocking. A Western-Blot with non-blocked antibody is normally used for comparison.

In the experiment you mentioned, they vary the principle a bit, as they are comparing two blots - one blocked with a succinylated library, one with a non-succinylated one. As the band disappears after treatment with the succinylated library, they confirm that their antibody is specific against succinylated proteins - at least in these three cases.

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  • $\begingroup$ So the the succinylated library blocks the binding of the antibody? $\endgroup$ – user2879934 Jun 9 '16 at 20:57
  • $\begingroup$ Right! And only the succinylated one, as the antibody does not recognise the Lys as such as you can see in the blocking with the non-succinylated library $\endgroup$ – aretaon Jun 9 '16 at 20:59
  • $\begingroup$ Why do it in the negative this way? Why not have an antibody that recognizes SucLys and then show that it's positive for the PTM and negative for the unmodified? $\endgroup$ – user2879934 Jun 9 '16 at 21:05
  • $\begingroup$ Or do such antibodies not readily exist? $\endgroup$ – user2879934 Jun 9 '16 at 21:05
  • $\begingroup$ I think its just an experimental problem: They have three proteins that contain SucLys and they have those libraries as they are commercially available. For a comparison as you propose, they would need a SucLys and a Lys version of their protein that would have to be (reliably) created beforehand. $\endgroup$ – aretaon Jun 9 '16 at 21:11

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