Why an antibody may detect tagged protein but not endogenous one and in some cases endogenous protein but not its tagged version?

We would expect that given antibody is going to recognize tagged and endogenous versions with the same intensity. However, over the last 25 years of working with antibody testing, we have seen on multiple occasions that a given antibody is detecting tagged protein but not endogenous one (Example 1), or it detects endogenous protein but not its tagged version (Example 2).

What can be the reasons behind it?

Example 1: Antibody detects only tagged protein
Depending upon the expression level, tagged proteins are most likely present in higher amounts as compared to an endogenous protein, especially of low expression, which can be checked in Protein Abundance Database.

Example 2: Antibody detects only endogenous protein
Detection of a target protein, depends upon an antibody binding to so-called epitopes, which are 2-15 amino acid long and can be either linear or conformational. Often times, in case of overexpressed proteins, steric hindrance caused by addition of a tag results in a different folding which may make antibody binding place not accessible. Other factors include: position of a tag (may influence the epitope antibody binds to), lack of post translational modifications, which are a part of antibody recognition.

Even though Western blot is done in denatured conditions, some proteins may refold following transfer to a membrane. In such situations using harsh denaturation conditions, like 6-8 M urea and drying a membrane in the air, following transfer may help to keep target protein unfolded as described here.

In summary: there is never a guarantee that a given antibody will detect both, native and tagged (recombinant protein).

Based on above, please keep in mind that a result showing that a given antibody is recognizing a tagged protein is never a proof of its reactivity to endogenous protein in analyzed cell extract.

A Antibody reactivity to tagged and recombinant protein
As seen in this example one and same antibody can detect recombinant protein with higer intensity, compared to endogenous not tagged protein. The reason is not equal abundance of both proteins.

Why an antibody may detect tagged protein but not endogenous one and in some cases endogenous protein but not its tagged version?

Technical tips

Latest

Archive