Surprisingly Easy To Use Biotinylated Polyclonal Against Amphiregulin

Autoimmune Genetics
KU Leuven
Senior Post-doctoral Scientist

Overall

Quality of Results

Ease-of-Optimization

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Company:

R&D Systems, a Biotechne brand

Product Name:

Mouse Amphiregulin Biotinylated Antibody

Catalog Number:

BAF989

Amphiregulin (Areg) is an EGF family cytokine produced by Treg, Th2 cells and other type 2 immune cells like ILC2. I investigated whether this biotinylated polyclonal could be useful for intracellular cytokine staining. It works quite well, with low background when appropriately titrated. Staining is specific as assessed by comparison with unstimulated cells. Any residual non-specific background can be reduced by co-incubation with goat serum or goat IgG (the anti-amphiregulin is goat origin).Staining is apparent on Treg, Th2 and ILC2, among other cell types.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Mouse lymphocytes

Primary Incubation

1h at 21C at 1:200, or 16h at 4C at 1:500

Blocking Agent

2.4G2 hybridoma supernatant, optionally also 5% goat serum

Secondary Incubation

Streptavidin BV605 (or any steptavidin) at 1:500 for 20min

Tertiary Incubation

None

Detection

BD FACS Symphony

Results Summary

On stimulated T cells or ILC2, the anti-Amphiregulin functions nicely, giving typical intracellular cytokine staining plumes. The separation of positive and negative is much better than expected for a polyclonal or a biotinylated antibody. A directly conjugated version would be superb.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Good, easy to use antibody against a less studied molecule. Color can be changed easily with strepavidin.

The Bad

Biotinylated polyclonal.

The Bottom Line

Quite good, flexible. Modest work required to set it up.

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