Choosing an epitope tag antibody: what a v5 antibody bought as an anti v5 antibody, a hemagglutinin antibody, an ha antibody bought as an anti ha antibody, an anti-ha tag antibody or an anti ha tag antibody, a myc antibody, a c-myc antibody, an anti myc tag antibody, a protein c antibody, a his tag antibody sold as an anti his antibody or an anti his tag antibody, a flag tag antibody, a gfp antibody or anti-gfp antibody and a bfp antibody each give you, why the ha tag antibody, myc tag antibody and v5 tag antibody reagents remove the reagent problem, and where on the protein to put the tag

Choosing the tag and placing it

1. Understand what the tag buys. One reagent, validated once, works on every tagged construct in the laboratory, in every application, with a known negative control in the untagged parent. That reproducibility is worth more than almost any improvement in an antibody against the native protein.
2. Choose between the common tags on practical grounds. The short tags differ mainly in length, charge and which reagents are best characterised for which application. Where two tags are needed on different proteins in one experiment, choose ones whose antibodies are raised in different hosts so the secondaries do not collide.
3. Decide the terminus from the protein's biology. A tag at a terminus that carries a signal sequence, a localisation signal or a processing site will disrupt it. Check both termini against what is known about the protein, and if neither is safe, an internal tag in a flexible loop is the alternative.
4. Use multiple copies when signal is the problem, and expect a cost. Repeating the tag increases the signal proportionally and increases the chance of interfering with folding, trafficking or interactions. Start with one copy and add only if detection genuinely fails.
5. Prove the tagged protein still behaves. Localisation, molecular weight, an activity assay and, where possible, rescue of the untagged protein's function. A tagged construct that has not been shown to behave like the original is a new protein you are now studying.

Tagging the endogenous locus rather than overexpressing

Knocking a tag into the native locus by genome editing keeps expression at physiological level and under native regulation, which removes the overexpression artefacts that plague transfected constructs. It costs an editing project and is usually worth it for a protein you will study for years.

It also gives an exact control: the parental line is the untagged negative, isogenic in every other respect, which is the cleanest specificity control available for any detection method.

Using the tag for purification as well as detection

Several tags have matched affinity resins, so the same construct serves detection, immunoprecipitation and purification. Elution by competition with free peptide is gentle and expensive; low pH elution is cheap and harsher on the protein.

Where the tag is used for purification, plan how and whether it is removed. A protease site between the tag and the protein is designed in at the start or it is not available later.

Multiplexing two tagged proteins

Use tags whose antibodies come from different host species, and cross-adsorbed secondaries. Two tags detected by antibodies from the same host require sequential detection and a blocking step, with its own controls.

Record which tag is on which construct in the plasmid map and in the cell line record. Tag confusion between two constructs in the same freezer is a recurring and entirely avoidable experiment lost.

Common questions

Why use an epitope tag antibody instead of one against my protein?

Because the reagent is already characterised and the same one works for every tagged construct. You also get a clean negative control for free in the untagged parent line, which is the hardest control to obtain otherwise.

Which tag should I choose?

Any of the well established short tags will do for detection. Choose on practical grounds: antibody host species where you need to multiplex, whether the tag's charge or length is likely to matter, and which reagents your laboratory already has validated.

Amino or carboxy terminus?

Whichever does not disrupt a signal sequence, a localisation signal, a processing site or a domain boundary. If both are problematic, an internal site in a flexible loop is worth trying, though it needs more validation.

Does tagging change the protein?

Sometimes, and that is why the control experiments matter. Show that the tagged protein localises correctly, runs at the expected size and retains function before trusting any result from it.

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