Secondary Antibody Resulted in Cross-Reactivity with Rat

Cell Biology-Immunology
Biotechnology Institute Thurgau
Graduate Student

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Company:

Jackson ImmunoResearch

Product Name:

Alexa Fluor® 488 AffiniPure Donkey Anti-Mouse IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Rb, Rat, Shp Sr Prot)

Catalog Number:

715-545-151

Clone Number:

Polyclonal

Info:Supplier Page View Product Specs

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I coated cover glasses with rat anti-MHCII antibodies and seeded dendritic cells on them to see the surface expression of several receptors within the engagement of MHCII by immunostaining. Unfortunately, I used this secondary antibody, sold to be minimal crosslinking with Rat, and it WAS CROSS-REACTING with my rat coating. I lost time and effort trying to work with this antibody, but nothing worked. I strongly do not recommend this antibody if you have to work with rat proteins.

Experimental Design and Results Summary

Applications

Immunofluorescence

Sample

Bone marrow derived dendritic cells (mouse)

Primary Incubation

None

Blocking Agent

BSA 5% tested (1h at RT)

Secondary Incubation

Phalloidin 647 1/250 + Donkey Anti-Mouse IgG (H+L)1/250 (1h at RT) 488

Tertiary Incubation

None

Detection

TIRF

Results Summary

Cross-linking of the secondary antibody onto the rat-antibody coated cover glass. I also tested to block with Donkey serum, even overnight I have got the same results. I used a fibronectin coating and an Armenian hamster antibody coating as controls. There was no sign of a cross-reaction then.

DOI or PMID #

N/A

Additional Notes

N/A

Image Gallery

Summary

The Good

Good fluorescence.

The Bad

Not specific.

The Bottom Line

Do not use it with Rat proteins.

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