Immunoblotting of Ubiquitin Phosphorylated at Ser65 in Cells

October 01, 2019

Dpt of Cell Bio
Havard Medical School
Postdoctoral Researcher

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Company:

MilliporeSigma

Product Name:

Anti-phospho-Ubiquitin (Ser65)

Catalog Number:

ABS1513-I

Clone Number:

Polyclonal

This antibody is used to probe for phosphorylation of ubiquitin in cells, a hallmark of PINK/Parkin pathway activation.

Experimental Design and Results Summary

Applications

Western Blot

Sample

NGN2-induced Neuron cells extracts

Primary Incubation

1:1000 overnight incubation at 4ºC, in 5% BSA diluted TBS+Tween (0.25%)

Blocking Agent

10% non-fat milk in TBS+Tween (0.25%) for 10 min

Secondary Incubation

1:20000, 1 h incubation at room temperature in 5% non-fat milk diluted TBS+Tween (0.25%)

Tertiary Incubation

Washed three times with TBS+Tween (0.25%) for 3 min each.

Detection

HRP-based chemiluminescent immunoblotting and x-ray film

Results Summary

This is able to detect endogenous levels of ubiquitin phosphorylation on Serine 65, a hallmark of PINK1 activation in cells. Signal is quite specific, despite few unspecific bands (as judge by the persistence of these band in PINK1 KO background).

DOI or PMID #

10.1016/j.molcel.2018.03.012

Additional Notes

N/A

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Summary

The Good

Good specificity across various cell lines

The Bad

Could be more sensitive. Need to long exposure times.

The Bottom Line

This an extremely useful antibody and one of the best on the market. I would highly recommend it to anyone studying Parkin/PINK1 pathway.

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