CD147 As A Measure Of ERAD

August 17, 2017

Food Science and Human Nutrition
Colorado State University
Graduate Student

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Company:

Abcam

Product Name:

Anti-CD147 antibody

Catalog Number:

ab64616

CD147 has been known to be a substrate of endoplasmic reticulum associated degradation (ERAD). The objective of this study was to find out if inhibition of ERAD would result in a degraded CD147. The first step was to determine whether inhibiting protein translation using cyclohexamide would reduce the production of CD147.

Experimental Design and Results Summary

Applications

Western Blot

Sample

H4IIE total cell lysates

Primary Incubation

1:1000 in 1:3 odyssey block:1X TBST (0.1% Tween) shaking, overnight at 4 degrees C

Blocking Agent

1:3 odyssey block:1X TBS at room temperature for 1 hr.

Secondary Incubation

1:15000 Goat anti-rabbit IRDye in 1:3 odyssey block:1X TBST (0.1% Tween) and 0.2% SDS for one hour at room temperature in the dark

Tertiary Incubation

N/A

Detection

Licor Odyssey Clx

Results Summary

This antibody works. There is a clear band around 55kDa and another weaker and around 42kDa. The problem is that CD147 is not inhibited using protein translation inhibitors such as cyclohexamide or emetine. CD147 may be a stable protein similar to structural proteins in H4IIE cells. However, at this point, we are confident this antibody works.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Clear band at 55kDa and very little non specific binding

The Bad

Antibody has been reported to show up at many different molecular weights ranging from 30-75. Always unsure if what we are looking at is the correct protein

The Bottom Line

There is a clear band at 55kDa using this antibody and Abcam reported this antibody showing up around 60kDa. May take a little optimizing to observe the various forms of the protein.

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