Assessing Function of Novel Protein Found on 2-D Gel Electrophoresis

[irfan_sadiq]

hello all,
if we have detected novel protein during 2-D gel electrophoresis,how we can see its function by using biotechnology or any other possible way.

[danfive]

Do you have the protein sequence (perhaps from trypsinization then LC-MS/MS) and subsequently the gene ID?
Obviously if you get gene ID you can go to making recombinant forms of the protein.
If not, then the peptide sequences from LC-MS-MS can be used to contract out antibodies. Find an antibody that will work for Immunopurification, then try isolating a large amount of the novel protein...then you can try LC-MS-MS (and getting gene ID, or homology match to known protein family), or Protein Crystallography or if you have a hunch on likely biochemical activity you can perform protein assays...

[nduong]

Can I apply the above method to 1-D gel electrophoresis?

[danfive]

Originally Posted by nduong

Can I apply the above method to 1-D gel electrophoresis?

Remember that bands in 1D gels can and usually consist of several if not dozens of proteins that happen to be the same MW. So you'll most likely be working with a mixture of proteins, not one single novel protein.

As long as the proteins in that band/mixture are present in the same amount then you can perform trypsinization and LC-MS-MS and get a decent list of proteins from that band. And like I said you'll have peptide sequences which can be synthetically made and used for creating unique antibodies. And from there you'll have to find the "right" antibody for something like Immunopurification....Also with enough hits (high confidence peptides) you may get the gene ID and go to recombinant genetics for amplifying the amount of protein....

The drawback is that it can backfire for rare proteins; they may not show up, because their signal or presence can and will be drowned out by the most abundant proteins...so the strength of the technology is to work with the most purified level of protein you can get.