protein binding

Using recombinant proteins to validate antibodies can provide false positive or false negative results. Your protein of interest is best observed in-situ in cell or tissue lysates.

Contributed by Mike Browning

It is important to look carefully at any antibody validation blots to be sure that a cell lysate and not simply a purified protein is being analyzed.

Antibody validation is a topic that has garnered a great deal of attention lately in discussions of the problem with the lack of reproducibility in science. One type of antibody validation that should be avoided uses purified recombinant proteins. In a typical study of this type a purified recombinant protein is run on a western blot and then the labeling of the new antibody is examined. Given that only a single protein is run on the blot, this “validation” study offers very little information about the specificity or sensitivity of the antibody. The blot obviously has no information about whether the antibody recognizes other proteins since no other protein is present in the blot. Moreover, absent any information about the relevance of the amount of recombinant protein used compared to the amount of endogenous protein in situ, the experiment does not even validate the ability of the antibody to bind to the protein of interest.

Thus a western blot with recombinant protein does little to answer the two key questions about antibody quality:

  1. Does the antibody possess the sensitivity to recognize the antigen in situ in a tissue of interest? 
  2. Is the antibody binding specific for the antigen of interest in situ?

Nevertheless, it is not unusual to see such data used to validate an antibody in product data sheets or even in refereed publications. 

A recombinant protein based assay can also sometimes falsely invalidate a phosphospecific antibody.

In the example above I described a situation in which a recombinant protein based assay was used to give a false positive validation. It is also not uncommon to see a recombinant protein based assay provide a false negative result i.e. to falsely invalidate an antibody. Such experiments are most commonly seen with phosphospecific antibodies. These antibodies can be extremely valuable tools as they permit one to evaluate the phosphorylation state of a single phosphorylation site on a specific protein.A critical question in the validation of such antibodies is whether they are indeed phosphospecific.A mutant recombinant protein with the phosphorylation site of interest mutated to a non-phosphorylatable amino acid is run along side the recombinant protein in a western blot. The antibody of interest is then tested for binding to this assay. Binding to the non-phosphorylatable mutant in such an assay has been used by some as evidence that the antibody’s phosphospecificity has been invalidated. Such data do not provide this evidence. A phospho-specific antibody will always have at least a finite affinity for the non phospho-site. Thus when, as is typical in this type of study, micrograms or hundreds of nanograms of the mutant protein are run on the blot, some binding is highly likely.

Incubating tissue in phosphatase is an easy way to validate a phosphospecific antibody.

In order to use a recombinant protein-based assay for validation it is necessary to do a very detailed dose response with multiple concentrations of both the mutant non-phospho and the phosphoprotein. Attention must also be paid to the concentration of the protein of interest in situ and also its level of phosphorylation at the site of interest.Determining these values is always quite problematic. Consequently the use of this validation technique is not recommended particularly when other much more relevant validation assays are available. The most common such assay is the western blot performed on lysates of the tissue of interest that had been incubated in the absence or presence of a phosphatase. In this assay the antibody is validated if it labels a single band in the control lysate and if the labeling is absent in the lysate that had been incubated in the presence of the phosphatase.

Non-phospho protein binding by a phosphospecific antibody needs to be addressed in the non-phospho column purification step.

One issue raised in the preceding paragraph was the fact that there is always a finite affinity of a phosphospecific antibody to the non-phosphoprotein. Such binding of the phospho-antibody to the non-phospho site can be quite problematic in negative affinity column selection of the phospho-antibody. In our experience we have often found that very good phosphospecific antibodies may sometime fail to flow through a non-phospho column. To avoid failures in such negative selection experiments it is very important to optimize the antibody to peptide ratio in using such a column. This issue will be discussed in more detail in a subsequent blog.

Further Reading:

SDS-PAGE Demystified – The Science Behind All Those Bubbles!

Antibodies in Western and IHC

Antibody Variability

Are Monoclonal Antibodies Really More Specific?

1% SDS is the Lysis Buffer of Choice for Most Western Blots

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Antibody specificityAntibody validationPhosphosolutions