Great and Versatile Anti-HA

April 06, 2016

Neurobiology
Harvard Medical School
Postdoctoral Fellow

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Ease-of-Optimization

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Company:

Cell Signaling Technologies

Product Name:

HA-Tag (C29F4) Rabbit mAb

Catalog Number:

#3724

We are investigating transgene expression in inner ear hair cells after adeno-associated virus (AAV) delivery. Transgene could have different tags, hemagglutinin (HA) tags is a widely used, small tag that usually does not interfere with protein functions. The specific goal of this experiment was to confirm subcellular localization of the transgene (i.e. hair bundle localization). We have tried different anti-HA antibodies, some of them did not work at all, this antibody seems very robust and specific even with a single HA tag on the transgene.

Experimental Design and Results Summary

Applications

Immunofluorescence

Sample

Organ of Corti explants, hair cells

Primary Incubation

1:250, 24-48h at 4C in 0.8% normal goat serum and 0.1% Triton X-100 in PBS

Blocking Agent

0.8% normal goat serum and 0.3% Triton X-100 in PBS

Secondary Incubation

Anti-rabbit Alexa 647 (or 594/488) 1:1000 in PBS, 0.1% Triton X-100, 8% NGS for 1h

Tertiary Incubation

NA

Detection

Alexa Fluor 647/594/488

Results Summary

Anti-HA staining confirmed that HA-TMHS (Tetraspan membrane protein in hair cell stereocilia (also known as: Lhfpl5 [Lipoma HMGIC Fusion Partner-Like 5]) correctly localizes to the stereocilia. It was also detectable in the cell body. Non-transduced hair cells do not show staining in the hair bundle and cell body. This is important, because antibodies are known to give some background in the hair bundle. This antibody used in this protocol yields great results.

Additional Notes

None

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Summary

The Good

Very specific, versatile.

The Bad

Sometimes have to be used at much higher concentration than recommended by the manufacturer.

The Bottom Line

This anti-HA antibody is really powerful in immunofluorescence, even for low protein concentrations. I have tried other anti-HA antibodies and this worked the best. It's also good for WB.

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