Bright and Reliable Secondary Antibody From Thermo Fisher

November 01, 2023

George Washington University

Overall

Quality of Results

Ease-of-Optimization

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Company:

Thermo Fisher

Product Name:

Mouse anti-Human Lambda Light Chain Secondary Antibody, APC

Catalog Number:

A15730

Clone Number:

The goal of this experiment was to titrate the correct amount of peptide and incubation time needed for the MHC presentation of the peptide on the cell surface.

Experimental Design and Results Summary

Applications

Flow cytometry

Sample

T2 cells (ATCC)

Primary Incubation

0.1uL Zombie Green viability stain in 100uL PBS, incubated at room temp for 15 mins

Blocking Agent

Human TruStain Fc block, 5uL diluted in 95uL FACS buffer (5% FBS in DPBS), incubated at 4C for 10 mins

Secondary Incubation

1 uL of TCR-like primary antibody diluted in 100 uL of FACS buffer (5% FBS in DPBS), incubated at 4C for 20 mins

Tertiary Incubation

10 uL of secondary antibody diluted in 100uL of FACS buffer

Detection

Flow cytometry in a beckman coulter cytoflex cytometer

Results Summary

This product helped me identify percent of cells positive for MHC presentation of my peptide, using an unconjugated TCR-like antibody as my primary antibody.

DOI or PMID #

N/A

Additional Notes

N/A

Image Gallery

Summary

The Good

Inexpensive and is very bright.

The Bad

Too bright at full test volume, no clear separation.

The Bottom Line

Good product if you need a validated secondary to stain a lambda light chain, and your primary is on mouse but do not use without adjusting voltage and titrating. As the image shows, most of my positive events were off scale.

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