Hi everybody,
Im about doing western blot and I've read that proteins must be denaturated for Western blotting.
I question how it can be possible for antibodies to detect my specific protein if it is unfolded??
Because your antibody will typically binds to a specific region of protein (epitope) , not to the whole protein. So if you denature or disturb the 3d form of your protein it wouldn't hamper antibody binding
Thank you, dear Subhankar Bose, for your response, but I think that if we produce an antibody against a folded protein, it may not attach to its unfolded form. Likewise, If the antibody is also attached to the protein's primary structure, it may bind to other unfolded proteins with similar epitopes in the mixture. So how western blotting can distinguish my specific protein among thousand of unfolded proteins in the mixture?
No, there is no need. It depends on the antibody, if it is recognizing the denatured and/or non-denatured protein. However, denaturing proteins (with SDS) for PAGE enables them being sorted by (apparent) molecular weight and running towards the positive electrode in the electrotransfer. So, if you intend to omit SDS, make sure you have blotting membranes on both sides of the gel.
You may look for a dot-blot assay in place of western because it omits the pre-fractionation of the protein sample by SDS-PAGE. PAGE will denature the protein for gel-based fractionation, if it is not needed, chromatographic fractionation and performing dot-blot will keep the protein in native (partially folded-intact) and improve the specificity.
Hello Ali Ghorbani
Since proteins are usually separated under denaturing conditions during gel electrophoresis in Western Blot, this will restrict the detection of proteins by antibodies recognizing structural epitopes in non-denatured proteins.
A key to understanding the varying performance of antibodies is to determine the binding sites, or epitopes, they recognize. Epitopes are generally divided in two categories,
1) linear epitopes where a stretch of continuous amino acids is sufficient for binding and
2) conformational epitopes where key amino acid residues are brought together by protein folding.
Conformational epitopes might be preferred for applications involving protein targets in their native state, such as therapeutic applications or flow cytometry.
On the other hand, linear epitopes might be preferred for applications in which the protein target is wholly or partially denatured during the sample preparation prior to the immuno assay, such as in Western Blot (WB), immunohistochemistry (IHC) or immunofluorescence-based confocal microscopy.
So, you should select an antibody that has been validated for Western Blot analysis. When choosing an antibody for Western Blotting experiments, the supplier/distributor provides comprehensive information relating to the type of antibody and its performance in multiple applications. If the chosen supplier/distributor provides validation data and assay conditions for Western Blotting, this is an indication that they recognize the importance of thorough antibody characterization.
Best.
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