IF of Chk1 pS345

Pharmacology
Boston University
Senior Post-doc

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Company:

Cell Signaling Technology

Product Name:

Phospho-Chk1 (Ser345) Rabbit mAb

Catalog Number:

2348L

Chk1 is phosphorylated on Ser 317 and Ser 345 by ATR protein kinases after DNA damage. Phosphorylation of Chk1 at S317 and S345 is a hallmark of activation of the cell cycle checkpoint. The UV-induced phospho-Chk1 S345 was examined using immunofluorescent microscopy.

Experimental Design and Results Summary

Applications

Immunofluorescence

Sample

HeLa human cancer cell line

Primary Incubation

Dilution 1:50 in PBS/0.05% Tween20/3% BSA blocking buffer. Overnight incubation at 4C

Blocking Agent

3% BSA / 0.05% Tween20 in PBS

Secondary Incubation

Cy3-conjugated anti-Rabbit 2nd antibody at dilution of 1:250 and 1hr incubation at room temperature

Tertiary Incubation

N/A

Detection

Fluorescent microscope (NIKON)

Results Summary

UV irradiated HeLa cells showed strong and clear Phosphorylation of Chk1 S345 signal, whereas control (undamaged) cells did not.

Additional Notes

The samples were fixed in 100% MeOH for 20 minutes at -20 degrees

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Summary

The Good

Very specific

The Bad

Need to use very high concentration (1:50)

The Bottom Line

Great antibody for IF study of phospho-Chk1 S345.

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