Replies

• 1

  John LaCava

  06 February 2009 | 05:33

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  Hi,
  There can be many ways to solve this question… however, the easiest (to my mind) is to use mass spectrometry (MS) and peptide mass fingerprinting (PMF). You could make an affinity purification resin by coupling your purified antibody to some stationary phase (agarose, sepharose, dynal.. to name a few…also, make sure your antibody will recognize the native protein, some Abs only work well for western or other assays). After exposing your sample to the resin, and washing it extensively, you should have significantly enriched and purified your sample. You can then elute the sample from the affinity column and prepare it by SDS-PAGE. The easiest way to elute for your purposes is probably directly into SDS-PAGE loading buffer. The sample thus separated on the gel can be visualized by Coomassie blue or silver staining (again, amongst others… make sure to use a mass spec compatible method). After staining, you can excise the bands from the gel and then follow a procedure for preparation for PMF. There is plenty of info out there. You will need access to core proteomic services or a collaborator able to carry out this procedure since it requires access to the MS machines and familiarity with MS database searches to interpret the MS spectra. The benefit of this approach is that not much material is needed (just enough to visualize by silver staining, for example… although a Coomassie band is preferable). The end result is confirming the identity of the unknown protein by virtue of its amino acid composition (and the unique masses thus associated). There are lots more details and things you can do… but this is how I would start.

  Hope this helps.
  John

• 2

  Sheldon Broedel, Jr.

  06 February 2009 | 15:57

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  Pooneh,

  We have made similar observations. The first thing we do is eliminate some of the obvious causes such as reactions to the secondary antibody or to partially reduced or denatured samples. We then verify the specificity of the reaction. Once we are confident of the specificity of the primary antibody, we identify the co-reactive protein using peptide sequence analysis.

  The results have revealed different reasons for the cross-reaction which have included a protein with the same epitope as the antibody (monoclonal in some cases), an isoform of the target protein and a non-denaturable aggregation of the target protein. Our approach has been to separate the sample by SDS-PAGE in two lanes, blot transferring the protein bands in both lanes and reacting one lane with the antibody. This is used as a reference to determine which slice of the transfer membrane to cut out for analysis. We send our samples to the Harvard Microsequencing Facility for peptide sequence analysis by MS/MS to identify the culprit. They have posted protocols on how to perform the sample preparation and do super work. We follow their recommendations to the letter and have never had a failure. Good luck.

  Sheldon

• 3

  pooneh Ardestani

  24 February 2009 | 09:21

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  thanks so much for your help :)