A Sensitive Antibody to Study Total Ubiquitinated Proteins in Cell Lysates

Biology
Seoul National University
Postdoctoral Researcher

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Company:

Cell Signaling Technology

Product Name:

Ubiquitin (P4D1) Mouse mAb

Catalog Number:

3936

Clone Number:

Monoclonal

This antibody detects ubiquitylated proteins in cell lysates with high sensitivity. I recommend using low % acryamide gel to probe high molecular weight proteins with poly-ubiquitin chains.

Experimental Design and Results Summary

Applications

Western Blot

Sample

HeLa cell lyate

Primary Incubation

1:2000 dilution, 4C overnight.

Blocking Agent

5% non-fat milk

Secondary Incubation

1:20000 dilution of anti-mouse IRDye-800

Tertiary Incubation

Washing with TBST X3, 5 min each

Detection

Odyssey

Results Summary

I confirmed this antibody using UBE1 inhibitor MLN7243. To resolve free ubiquitin, I would use high % acrylamide gel. I used NuPAGE-bis-tris gel 4-12% with MES buffer, which gave nice ubiquitylated substrate bands. However, the free ubiquitin band appeared as smear at the bottom of the blot.

DOI or PMID #

N/A

Additional Notes

N/A

Related Categories

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Summary

The Good

Good quality

The Bad

Nothing

The Bottom Line

Sensitive antibody for total ubiquitylated substrates.

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