It is common to include a small segment of the viral hemagglutinin (HA) coat protein (typically with the amino acid sequence: YPYDVPDYA) in gene expression vectors as an epitope tag. The segment is chosen due to its high immunogenicity and immuno inaccessibility in the native hemagglutinin conformation, as indicated in the original publication [2]. Hemagglutinin antibodies can easily recognize such a tag, and the expressed protein can then be purified or characterized. Multimeric HA tags, such as 3 copies of HA in a row (HAx3), YPYDVPDYAGYPYDVPDYAGYPYDVPDYA, can significantly improve the affinity toward its cognate antibodies. For example, Roche (now MilliporeSigma) 3F10 clone anti-HA antibody has Kd of 0.38 nM for HA and has 0.067 nM for HAx3 under immunoprecipitation conditions [3]. HA tag is one of most commonly used tag systems. Other commonly used ones include c-Myc and His6. Schüchner S et al suggested that clone 12CA5, raised against a 36-residue peptide from hemagglutinin, might display context-dependent affinities, based on their results with myc tag antibodies [4].

In conjugation with an HA hemagglutinin tag antibody, proteins tagged with the HA segment, or even in a "spaghetti monster" fashion [5], can be easily identified in a Western blot or immunocytochemical experiments, or in live cell imaging, eliminating the generation of any protein-specific antibodies. Thus, HA hemagglutinin tag antibody is one of most commonly used antibodies in protein expression and cell biology studies. A typical example is indicated in Figure 1.

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Figure 1. A and B. Immunostaining of S2 cells transfected with HA-tagged Rab11 (red). C. S2 cells transfected with FLAG-tagged Crag and/or HA-tagged Rab11, Rab11-DN, or Rab11-CA were harvested and lysed, detected by the anti-HA antibody. Anti-HA gel was used to pull down HA-tagged proteins, which also co-immunoprecipitated the FLAG-tagged CRAG. Images from [1].

To facilitate Labome visitors to locate the most suited antibodies, including HA tag antibody, Labome surveys randomly selected publications. Labome has reviewed formal publications citing HA antibodies. Table 1 lists the major suppliers.

HA antibodies have been employed in western blot [9, 13], immunoprecipitation [9, 18], immunocytochemistry, immunohistochemistry, ELISA, and chromatin immunoprecipitation (Table 2).

More in-depth discussions of HA antibodies based on selected publications from selected suppliers are as follows. A few examples are listed here for those not discussed separately. Morishita R et al examined the cross-reactivity of MBL anti-HA antibody clone TANA2 with a human protein array (CF-PA2Vtech), and found it recognized 9 human proteins in addition to the HA tag, all of which contained the epitope sequence PDY [20]. Feldman D et al used rabbit anti-HA monoclonal antibody (clone C29F4) from CST to screen HeLa-TetR-Cas9-FR cells in situ (immunocytochemistry) and through FACS [24]. Zhao N et al grafted six CDRs from 12CA5 scFv into an anti-H4K20me (clone 15F11) scFv scaffold to develop frankenbody, a fast and versatile live cell imaging probe based on HA [5].

Santa Cruz HA antibodies are listed here. Götzke H et al utilized Santa Cruz anti-HA F-7 antibody (sc-7392) in Western blot to compare different protein tags [13]. Mouse monoclonal Santa Cruz Biotechnology anti-HA antibody was used to perform western blot assays to investigate the role for the Ras/Epac2 pathway [26] and the role of MMS19 [27]. Its monoclonal HA antibody was also used in immunoprecipitation [28]. Santa Cruz Biotechnology rabbit anti-HA (Y-11) was used to perform ChIP assay [29] and immunofluorescence [30].

Abcam HA antibodies are listed here.

Abcam rabbit polyclonal anti-HA antibody was used to perform immunocytochemistry to investigate myelin membrane assembly [31] and immunohistochemistry to study Vibrio cholerae biofilms [32]. Its FITC-tagged anti-HA antibody was used at 1:200 to perform immunocytochemistry to investigate innate immune response against Listeria [33]. Abcam anti-HA antibody was used in Western blot [21], immunocytochemistry [34] and immunoprecipitation [35].

Rockland polyclonal rabbit anti-HA antibody was used to perform immunohistochemistry [36] and in Western blot and immunocytochemistry [37].

Laflamme C et al used anti-HA magnetic beads from Thermo Fisher Scientific (cat# 88837) to isolate lysosomes from HEK-293 cells transfected with Tmem192-3xHA (Addgene #102930) and to isolate mitochondria from those transfected with 3xHA-eGFP-OMP25 (Addgene #83356) [17]. Huang H et al used HA-conjugated magnetic beads (88836) from Thermo Fisher Scientific to IP HA-conjugated proteins from Hela cells [9].

Molecular Probes rat anti-HA antibody was used to perform immunocytochemistry assays to study the interaction mode between Hox transcription factors and PBC class proteins [38].

Sandstrom A et al used MilliporeSigma rat anti-HA (Roche, 3F10) for immunocytochemistry at 0.5 μg/ml for western blot at 100 ng/ml [22]. Zhao N et al detected 4 × HA-mCh-H2B and 4 × HA-mCh-β-actin transiently expressed in U2OS cells with anti-HA antibody 12CA5 (MilliporeSigma Cat # 11583816001) at 0.5 ug/mL in Western blot [5].

Many suppliers provide anti-HA antibodies which have been cited by multiple publications. Generally speaking, monoclonal anti-HA antibodies are preferable over the polyclonals, and the above survey results list some of the more cited suppliers.

One consideration is the price and the dilution of the antibody stock for a specific application. Since anti-HA antibodies tend to be used in multiple experiments, by many researchers in the same lab, it is also important to consider the dilution factors of the antibodies provided by different suppliers. For example, an anti-HA antibody with 1:5000 dilution is worth ten times of another anti-HA antibody with 1:500 dilution for the same applications.

Several reasons can account for this: 1) the early termination of the translation of HA-tagged protein; 2) non-specific anti-HA antibody, or anti-HA antibody is polyclonal; 3) partial degradation of the HA-tagged protein; 4) the concentration of anti-HA and/or the secondary antibody is too high.

For negative control, use an unrelated antibody, or control with the same host species, class, and subclass as the anti-HA antibody, as the immunoprecipitating antibody. For positive control, use an expression vector with only the HA for the transfection.

Likely the target protein was not tagged by HA, or HA-tagged protein is degraded.