1
$\begingroup$

What are the alternatives to using a histidine-tag in detecting protein-protein interactions? Why would they be more beneficial than his-tags?

$\endgroup$
1
  • 1
    $\begingroup$ Histidine tags on proteins do not inherently enable the detection of protein-protein interactions. Histidine tags allow one to attempt to purify your protein of choice using affinity chromatography on resins that have been substituted with chelated Ni++ (as I recall). There are two potential advantages to using this approach. 1st, you may be able to elute any bound proteins using a buffer containing histidine (i.e., non-denaturing conditions). 2nd, If your protein is insoluble you can solubilize it with urea and/or SDS, and the protein will still bind the affinity matrix. $\endgroup$ – mdperry Nov 29 '15 at 22:15
2
$\begingroup$

This depends on the technology you're using for the interaction detection. Surface Plasmon Resonance and Isothermal Titration Calorimetry (and others) don't require tagged proteins of any kind. Though it doesn't hurt either one.

If you're looking purely at chromotography, any tag that will stably capture. I commonly see GST, FLAG, and MBP in order of occurrence.

$\endgroup$
1
$\begingroup$

The His tag is part of a group of small peptides known as epitope tags. They vary in length from 5 or 6 amino acids up to 10-15. Some common ones include HA, FLAG, Myc, and V5. Once common element they share is that the sequences are quite immunogenic, so it is relatively straightforward to raise antibodies to them. They are also soluble, so they don't affect the tagged protein's structure much. This is not always the case in practice, though.

These tags are mainly used in studying protein-protein interactions by antibody-based detection methods - affinity chromatography, Western blot, co-immunoprecipitation, immunofluorescence, etc. They are extraordinarily useful for cases where the tagged protein does not have a good antibody available, or you are doing multiple parallel experiments with many tagged proteins and you don't want to optimize the conditions for each protein-specific antibody individually.

There are various reasons for using another epitope tag over a 6X or 10X His tag, the most common being the His tag is interfering with the proteins structure, function, or binding characteristics somehow. This can sometimes be solved by moving the tag from the amino- to the carboxy-terminus of the protein, if possible, but choosing a different tag is a common strategy. Another purely practical reason to use a different tag is that one protein in your experimental system may already be His tagged, so you need something different. Finally, the conditions of your experiment may be such that a His tag cannot be used, such as harsh elution conditions requiring the denaturing of the protein.

$\endgroup$

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.