Very potent antibody for pERK flow staining

November 24, 2014

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Microbiology and Immunology
Cornell University
Research Associate

Company:

Cell Signaling

Product Name:

Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10) Mouse mAb (Alexa Fluor® 488 conjugate)

Catalog Number:

4374S

Upon T cell receptor activation, T cells turn on MAPK pathways including the ERK (p44/42) activation, which is critical for T cell activation and further differentiation of effector functions. Given the complexity of immune cell populations, flow cytometry is normally required to distinguish cell types to pinpoint cells that are up-regulating ERK activity ex vivo. The reviewed product from Cell Signaling turned out to be the best in the reviewer's hands in giving a robust signal. We compared this to another antibody and concluded that this product is a potent indicator for pERK signal obtained through flow cytometry.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Mouse splenocytes

Primary Incubation

Ex vivo

Blocking Agent

1% BSA/PBS

Secondary Incubation

NA

Tertiary Incubation

1:100, 40 mins in room temperature

Detection

LSRII

Results Summary

T cells stimulated with PMA for 5 mins exhibited strong pERK signals detected by this antibody; while antibody from a different vendor only gave very moderate signals. Negative control was the unstimulated cells stained with the reviewed product.

Additional Notes

Cells fixed in 4% PFA in 37C for 10 mins, permeabilized in 100% methonal on ice for 10 mins, and stained in 1% BSA/PBS with anti-CD3 and indicated pERK antibody for 40 mins.

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Summary

The Good

Strong signals, high specificity.

The Bad

None

The Bottom Line

Proper stimulation is critical for inducing ERK phosphorylation, thus when stimulating T cells through TCR activation, positive control with PMA stimulation is important to make sure the antibody is working.

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