Help on protein integrity Western Blot - Sample preparation and detection (May/06/2005 )
I am relatively new to the field of immunoblots and immunoassay and have some questions i hope to find answers to.
Our current practice is to run a SDS-PAGE prior to blotting. However, I was told by a fellow colleague recently that I should have run a native gel to "preserve the antigenicity" of the protein. However, I could not find information to back up the reason for using SDS-PAGE or why it is preferred over Native PAGE.
My query is, does it matter if the protein was denatured? Would it affect the affinity of the antibody for the protein epitope?
Does the antibody recognize only the amino acid sequence?
I would greatly appreciate it if anyone can point me to a link or any articles detailing the principles of antibody binding on blots. Thank you very much!
Denaturing you protein on SDS-PAGE will expose more epitopes on the protein and thus provide better chances to detect a protein witha polyclonal or monclonal primary antibody after being separted on the dnaturing gel according to its molecualr weight and charge. The native gel seprate proteins acoording to their charge , and since proteins are mostly availble in complexes so your protein might be covered by other proteins and it s migration on the gel is affected emmnsly, and thus its epitopes (antigenic comparment of the surface of a protein in its 3D confirmation) are not exposed and results in the lack of the detction of your protein. In summary, the SDS-PAGE dnaturing gel is the way to go. Good Luck!
Thank you very much for your help. That clears things up!
Just one more issue. When u mention epitope, does it mean that the epitope is present irregardless of whether the protein is denatured thus losing its 3D conformation? Cos i was under the impression if the epitope unfolds, the antibodies cant bind.
Usually when we raise polyclonal antibodies, we get the pure form of the protein, then run it on an SDS-PAGE gel and then elute that band concentrate it and thenuse it to inject ina rabbit or what ver is the host animal to generate antibodies. This process results in denatartion of the protein, what this means is that some epitopes might be denatured, however, the intrinsic epitopes are frutehr exposed and thus their 3D is not affected, which antibodies are generated against, thus having binding specificity against the taregt antigen (protein in this case) in ELISA or WB. I hope this helps, good luck!
Thank you for your help. Have a great day!
This might not be the whole story though. There are quite some antibodies out there which are conformation specific. So once the protein is denatured in SDS-PAGE, these antibodies won't pick up the epitope by Western anymore. For same reason, some antibodies only IP or ELISA but not Western. So it depends on what kind of protein/antibody pair you're looking at although SDS-PAGE is used more frequently in many labs because the conformation-specific antibodies are far more difficult to make than sequence-specifi antibodies, thus far more rare.
this sounds difficult but may i know if there is any ways to identify if the antibody is sequence or conformation specific without using the proteins to check? Or how is it usually done in labs? I am not well versed in this aspect. Thanks for your help!