Phospho-SAPK/JNK (Thr183/Tyr185) (G9) Mouse mAb Antibody Review

March 08, 2016

None
Institute of Protein Biochemistry, Italian National Research Council, Napoli
Postdoctoral fellow

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Company:

Cell Signaling Technology‎

Product Name:

Phospho-SAPK/JNK (Thr183/Tyr185) (G9) Mouse mAb

Catalog Number:

#9255

We are interested in studying a protein that we hypothesized to activate JNK when it is expressed in the cell. We transfected this protein in an inducible vector. When lysates of induced and un-induced were processed for western blotting we see the JNK activation by using Phospho-SAPK/JNK (Thr183/Tyr185) (G9) Mouse mAb (#9255). We concluded that induction expression of this particular protein of interest activates JNK pathway.

Experimental Design and Results Summary

Applications

Western Blot

Sample

Hela Cells

Primary Incubation

1:1000 in TBS-T with 5% nonfat dry Milk, incubated O/N at 4°C

Blocking Agent

5% Nonfat dry milk in TBS-T for 1hr

Secondary Incubation

Incubate membrane with Anti-mouse IgG, Peroxidase-linked Antibody (401215-MerckMillipore, 1:8000 diluted) in blocking buffer with gentle agitation for 1 hr at room temperature.

Tertiary Incubation

None

Detection

Added Luminata Crescendo Western HRP substrate (WBLUR0100) on top of the blot for 3-5 min, wiped off excess solution from the blot and wrapped securely in a plastic wrap. Exposed the blot to x-ray film for 30 seconds -5 min.

Results Summary

Expression of the protein of our interest activates JNK pathway.

Additional Notes

None

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Summary

The Good

Detects the Phospho-JNK without background if you block with non fat dry milk as blocking agent.

The Bad

If you use BSA for blocking, there will be light nonspecific detection of some other bands.

The Bottom Line

Good antibody for the detection of activated JNK pathway.

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