Flow Cytometric Detection of Biotinylated Antibodies

September 01, 2015

Microbiology and Immunology
University of Miami
Post-doctorall fellow

Overall

Quality of Results

Ease-of-Optimization

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Company:

BD Biosciences

Product Name:

Streptavidin BV711

Catalog Number:

563262

Our lab is concerned with studying transcriptome and signaling changes that occur during transitional B-cell development. This is a major checkpoint in peripheral tolerance where autoreactive B-cell clones are purged. For this particular study, kappa and lambda light chain ratios were examined in splenocytes. Lambda light chain was detected using a biotinylated antibody followed by Strepatividin BV711. This secondary step provided additional amplification of lambda light chain signal and had minimal spectral overlap with BV786.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

C57BL/6 splenocytes

Primary Incubation

15-20min in FACS buffer 1:200 biotinylated antibody

Blocking Agent

Fc block 5min prior

Secondary Incubation

15-20min in FACS buffer 1:300 final concentration for SAV BV711

Tertiary Incubation

NA

Detection

Flow cytometry

Results Summary

BV 711 Streptavidin conjugate detected lambda light chain antibody conjugated to biotin. This antibody has minimal spectral overlap with BV786 but should not be used with Alexa Fluor 700.

Additional Notes

None

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Summary

The Good

SAV BV711 provided great detection of biotinylated antibodies

The Bad

SAV BV711 concentration must be optimized for each biotinylated antibody used

The Bottom Line

This is a very good streptavidin conjugate to use for most fluoroschrome combinations

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