Active p38 alpha Blotting

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

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Company:

Cell Signaling Technology

Product Name:

Phospho-p38 MAPK (Thr180/Tyr182) Antibody

Catalog Number:

9211

Many signals via MKK3, MKK6, activate p38 alpha MAPK by phosphorylation at Thr180 and Tyr182, which in turn phosphorylates a many other proteins.The levels of Thr180 and Tyr182 phosphorylated p38 alpha is used as a measure of activation of stress activated MAPK pathway.

Experimental Design and Results Summary

Applications

Western Blot

Sample

HePG2 cells

Primary Incubation

Primary antibody (1:1000) in dilution buffer (TBST with 5% BSA) with gentle agitation overnight at 4°C.

Blocking Agent

TBST with 5% w/v nonfat dry milk

Secondary Incubation

Incubated the membrane with the Goat Anti-Rabbit IgG, H & L Chain Specific Peroxidase Conjugate (Cat No: 401315) 1:10000 diluted in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.

Tertiary Incubation

none

Detection

Incubated the membrane with appropriate amount of Luminata Crescendo Western HRP substrate (WBLUR0100 Merckmillipore) for 5 min at room temperature. Drained the membrane of excess developing solution (without letting it to dry) wrapped in transparent plastic sheet and exposed the x-ray film for 1 min.

Results Summary

Hydrogen peroxide treatment to cells is a well known activator of p38 alpha and downstream pathway . The antibody detects the activated p38-alpha when treated with hydrogen peroxide to the cells.

Additional Notes

This antibody is also used in the published article PMID: 26660341

Related Categories

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Summary

The Good

Works well without detecting much background or unspecific proteins.

The Bad

None

The Bottom Line

Useful antibody for determination of p38 pathway activation.

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