What does it mean when an antibody company says an antibody “does not work” in western blots?
I started wondering about this question the other day after a colleague told me about a company that “validates” all of their antibodies by immunostaining in no less than 5 different tissues!  We examined the company’s website and in some cases western blots (WB) showing labeling of a single band were also presented.  But for the vast majority of products on the site only the apparently innocuous phrase “does not work in WB” was seen.  What does this mean?  I think for many antibody users (especially people who do not do WB and use various immunostaining protocols like IF or IHC) this failure to “work” in WB is often interpreted as meaning something akin to “don’t worry about the WB data, see if the antibody ‘works’ in immunostaining.”  However, I think any user of an antibody that “does not work in WB” should be very worried indeed.  This is because in the overwhelming majority of cases “does not work in WB” means the antibody labels many different proteins in a WB.  It is extremely rare to get an antibody that labels nothing in a WB. 
Sap102 and THRAB_edited-2 
Examples of antibodies that do and do not work in WB are shown in Fig. 1.  The lane at the right shows staining of a brain lysate with an antibody raised against tyrosine hydroxylase (THRAB) and it shows labeling of a single band of Mr 60,000 which is the approximate Mr of TH.  The lane at the left shows staining of an antibody that does not work in WB.  The antibody shown was raised against a protein Sap 102.  As can be seen in the image of the WB, the antibody does recognize a band at the appropriate Mr for Sap 102, but the antibody also recognizes many other bands as well.  This antibody ”did not work in WB” and it was thrown in the trash where it belonged.
So remember when you read the words “does not work in WB” you should translate that as “this antibody probably cross reacts with a number of different proteins.”

Summary
Antibodies That DO NOT WORK in Western Blots
Article Name
Antibodies That DO NOT WORK in Western Blots
Description
When an antibody company describes one of their products as not working in western blot, this doesn't necessarily mean that no banding was observed. In fact it may mean that many contaminant bands were visible. This is a huge concern when considering the antibody for applications such as IHC and IF.
Author
Mike Browning
Publisher Name
PhosphoSolutions
Publisher Logo

5 thoughts on “Antibodies That DO NOT WORK in Western Blots

  1. Hi Mike,

    Stumbled across your blog -really interesting. I'm currently writing up my PhD thesis and going through A LOT of western blots and IF images. I don't know if this is a stupid question or not, but do you know why some antibodies don't work for IF? (or at least don't work well)

    thanks,

    Lex

  2. Hi Lex,

    I apologize for not answering sooner but I was on vacation with my family.

    First, let me emphasize that I am no expert on IF or IHC so I will first refer you to some resources for detailed answers to your questions. The site Linkedin.com has a number of discussion groups that address this issue. For example: http://tinyurl.com/lyblf83
    http://tinyurl.com/m747onp and also http://tinyurl.com/l3czt6n
    If you are a member of LinkedIn or join it you can start a discussion with you own specific issues if you like.
    Having said that I know there are a number of reasons why an antibody may not work in IF or IHC. Let’s assume you are using an antibody that has been validated with a western blot (WB) and the antibody labels only a single band at the appropriate molecular weight in the blot. When such an antibody does not work in IF or IHC there are a number of possible explanations. Two that readily come to mind are described below:
    1. One common issue is that proteins in a WB are linearized by treatment with SDS and reducing agents. This affects the structure of the protein and antibody binding. In contrast in IF or IHC the proteins are cross linked with the fixative used and this also can affect the structure of the protein. It may be that the antibody can access its target epitopes in the protein in WB but cannot access the protein in IHC because its structure is different due the fixative or because the antibody simply cannot penetrate the tissue to gain access to the protein. Techniques referred to as “antigen retrieval” can address this issue. Some refs: http://www.ncbi.nlm.nih.gov/pubmed/17471119 http://www.ihcworld.com/_intro/antigen-retrieval.htm
    2. It may be that the antibody “fails” not because you did not get a signal but rather you got a signal in tissues where the protein is thought to be absent e.g. in a of knockouts. Thus even though the antibody labeling is specific in WB it seems to be non-specific in IHC as it labels something other than the target protein. It may be that the antibody did not react with this “non-target protein” in WB because it was present in too low a concentration to be detectable in whole cell lysate. However the “non target protein” in concentrated in certain cellular locales such that your antibody does show labeling in a tissue slice in IHC. It is also possible that this off target labeling is from using to high a primary antibody concentration and adjusting your incubation conditions may help.

    I have gone on far too long about a topic with which I have too little experience. I am sorry if I haven’t been more help. Do try the resources I mentioned above. Good Luck.

  3. Hi Mike,
    I just came across your blog when googling my doubts about antibodies, and found a lot of very interesting information!
    I’m a PhD student working on a project combining IHC and WB againts a certain protein in samples from both mice and sheep. We first performed IHC on these samples and found a distribution of the protein that was somehow different in both species. The surprise came when WB was performed (with the same antibody) and no bands were observed in the sheep homogenates. What would you thing of an antibody that “does not work on WB” because no bands can be seen? Would you use it for IHC? We really don’t thing that the antibody’s staining in IHC is an artifact although it is true that we keep getting a lot of background.
    Many thanks in advance!

    Tomas

  4. Hi Tomas,
    When you get no signal in WB one should be careful about using the ab in
    IHC. This is because w/o WB you have NO information about the specificity
    go the ab. This does Not mean the ab can’t work in IHC just that you
    currently have NO data on whether it is working specifically. An antigen
    block is not at all helpful here despite the fact that it is a commonly
    used technique. In my opinion you need some other data to show specificity
    before claiming your IHC result.
    Mike

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