Anti-RFP Antibody For Flow Cytometry

April 04, 2018

BlueskyReddit
Autoimmune Genetics
KU Leuven
Senior Post-doctoral Scientist

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Company:

Rockland Immunochemicals

Product Name:

RFP Antibody Pre-adsorbed

Catalog Number:

600-401-379

RFP is frequently used as a reporter protein. While bright and easily detectable by flow cytometry and imaging, expression is usually lost or reduced when the plasma membrane is damaged. If the cells need to be fixed or fixed and permeabilized for analysis of intracellular components (e.g., transcription factors), the RFP signal becomes too weak to be useful. I investigated the use of this anti-RFP antibody for flow cytometry, focusing on whether it could recover RFP signals in cells that had been extensively permeabilized.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Mouse splenocytes

Primary Incubation

1:2000 in eBioscience Foxp3 permeabilization buffer at 4C for 16h.

Blocking Agent

2.4G2 hybridoma supernatant

Secondary Incubation

None (I conjugated the antibody with Lightning Link PE)

Tertiary Incubation

None

Detection

BD FACSymphony with 561nm laser

Results Summary

The antibody works well. RFP signal can be largely restored after near-complete loss. Minimal non-specific binding is observed on splenocytes that do not express RFP when the antibody is used at a dilution of >1000.With the antibody directly conjugated to PE, normal rabbit serum could be used to prevent non-specific binding, and the anti-RFP could then be used less diluted. The antibody also works well with a secondary anti-rabbit. In this case, quenching of the original RFP signal will permit the user to redirect the signal from the RFP into a different fluorescent channel if desired (e.g., by using anti-rabbit AF647).

DOI or PMID #

N/A

Additional Notes

N/A

Image Gallery

Summary

The Good

Highly specific, works when diluted well, and rescues "quenched" fluorescence.

The Bad

Requires user conjugation (Lightning Link) or a secondary.

The Bottom Line

This antibody permits analysis of RFP signals in fixed and permeabilized cells.

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