Hi, does anybody know if it is possible to use an IHC antibody for WB? the information sheet of the product doesn't mention if it was tested for WB previously.
Thanks in advance
In SDS-PAGE, the proteins are denatured, so there is a chance that an antibody that works in IHC on rather native conformation does not recognize the target in Western Blots.
Have you searched the literature if there are any publications where this antibody has been used successfully for WB? Otherwise, only an experiment will tell you if it works or not.
Alternatively, look for an antibody that has been shown to work in WB, there should be plenty of anti-p53 out there, this protein is quite frequently researched.
Hello Rizki
In Western Blot, the proteins are linearized due to the treatment with SDS and reducing agents. Because of this treatment the protein structure and antibody binding are affected. On the other hand, in IHC the proteins are cross linked with the fixative that is used which will also affect the structure of the protein of interest. So in both techniques the treatments differ.
The antibody meant to be used in IHC will get access to the protein because the structure of the protein differs according to the treatment provided and this antibody has been validated by the company for IHC use only.
If the same antibody is used in Western Blot wherein the protein is denatured, a broader range of epitopes will be made available for binding by the antibody. So, you will get many non-specific bands on the blot if you use antibody meant to be used for IHC.
The data sheet of the antibody always mentions as for IHC use only, or for WB use only, or for both IHC and WB use. So, you cannot use IHC antibody for Western Blot.
I hope this is helpful.
Best Wishes.
Wolfgang Schechinger a paper cited on the webpage of the product did use the same antibody for WB. However, that paper provided no further detail on the dilution.
I really wish I could get a new antibody but my situation does not allow me to. rough time.
Thanks for your input anyway, I appreciate it
Malcolm Nobre Thank you for the input. I was hoping if I could make this antibody to use because the webpage mentioned that this antibody had been used for WB in a paper. But the paper in question did not give further detail
Rizki, in that case, begin with a rather high concentration (e.g. 1:1000, the antibody seems to be unpurified ascites), you always may re-use the solutions (at least during assay development) and dilute further (use 1% BSA in PBST as diluent, or a similar concoction) to minimize antibody usage.
Or begin with 1:10.000 and see where you get, you could add more antibody if you don't see the desired signal or dilute further if signal and esp. background are too high.
Do the test incubations with small membrane strips in sealed tubes on a slow rotator or by occasional manual agitation. If possible, have a positive and a negative control on the strip. Add a little (0.1% m/v) sodium azide to prevent microbial growth. So you can do a lot of optimizations with little waste of antibody.
If you can use an antibody tested for IHC also for WB depends on the recognized epitope. The fixation with formalin crosslinks the proteins in a more native state but can have an influence on the accessiblity of the epitope(s), which is reversible to some degree by epitope retrieval. In WB the proteins are normally denatured and therefore not all epitopes might be present under this condition.
You can always test an IHC antibody in WB and check if it works. I suggest that you try several dilutions (around 10x less concentrated than for IHC).
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