What are the advantages of Epitope tags?

 

 

What are the limitations of Epitope tags?


A few can be as follows

 

 

How do I introduce the tag?


The two standard approaches to tagging a cloned gene are: (a) An epitope encoding oligonucleotide is inserted into the coding sequence, or (b) the coding sequence is inserted into an expression vector that already carries the epitope tag. BioLegend does not provide cloning vectors or oligos for cloning. The cloning protocols can be found online or in the literature. When tagging by oligonucleotide insertion, it is important to take into account codon usage preferences for the target cell or organism.

 

 

Will the epitope tag interfere with the function of my protein?


That has to be determined empirically. However, it is possible that the insertion of the epitope tag may interfere with protein function.

 

 

Where should I put the epitope tag?


In the literature, the tag has been placed at, or very near, the extreme N or C terminus of the target protein. There are a few reasons behind this choice. Some are historical-like the first proteins to be epitope tagged were tagged at the termini, and when new proteins were tagged it seemed wise to do what had worked in the past. Some of the reasons are practical-tagging is often performed using expression vectors that automatically put the tag at a terminus. And some reasons are theoretical-termini are frequently chosen in the belief that proteins will tolerate additions more readily at these locations than at other sites. While the latter belief may be true-termini are rarely included in active sites, for example-it is also true that many proteins are known for which terminal sequences are critical for function. The termini also appear favorable because they are likely to be on the outside of the folded polypeptide, where one wants the tag to go, and not in the hydrophobic core. But it must be remembered that, due to simple geometry, most of the amino acids in any protein are on the outside, and so, if a protein is tagged at a randomly chosen site, the tag will probably wind up on the outside anyway.

 

 

Why is there no signal with the epitope tag antibodies on a western blot?


A lack of signal following western blotting may indicate a few different problems.

  1. No or very poor transfer. This can be addressed by quickly checking the membrane with Ponceau-S staining.
  2. The expression level of the GFP-tagged protein may be too low. Load more ug quantity of the sample and include a positive control.
  3. A very diluted antibody may be the problem, try a few different concentrations of the antibody to probe the western blot.
  4. Also, a remote possibility is that the GFP tag is out of frame and is not expressed resulting in a lack of any signal.

 

 

Proteins can be detected with an anti-protein antibody, but not with the epitope tag antibody. What's happening here?

It is possible the target protein was not tagged, the tag is out of the reading frame, or the protein is degraded.

 

 

How to set up controls for co-immunoprecipitation experiment?


For negative control, use an unrelated antibody, or control with the same host species, class, and subclass as the immunoprecipitating antibody. For positive control, use an expression vector with only the epitope tag of interest.

 

 

During Western blot with epitope tag antibodies, there are multiple bands.


Several reasons can account for this:

  1. the early termination of the translation of epitope-tagged protein;
  2. non-specific detection with anti-epitope tag antibody due to no/poor blocking of the immunoblot.
  3. partial degradation of the protein
  4. the concentration of the primary or the secondary antibody is too high.
  5. Washing between the incubations is not efficient

 

 

What applications can I use epitope tagged antibodies for?


The commonly used applications are western blotting, immunoprecipitation and protein purification. These can also be used for immunofluorescence microscopy and flow cytometry.