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? and 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. So 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.
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. Such 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 interested mutated to a
non-phosphorylatable amino acid is run along side the recombinant protein in a
western blot. The antibody on 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
such evidence. A phospho-specific
antibody will always have at least a finite affinity for the non phospho-site. Thus when, as is typical in such studies,
micrograms or hundreds of nanograms of the mutant protein are run on the blot,
some binding is highly likely. In order
to use such an 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 such a validation
technique in 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 such an 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.
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-phosphosite 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.
