Detection of HSP47 Knockdown using siRNA in NIH3T3

February 13, 2015

Biomedical Engineering
Oregon Health and Science University
Graduate Research Assistant

Overall

Quality of Results

Ease-of-Optimization

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Company:

Novus Biologicals

Product Name:

Hsp47 Antibody (M16.10A1)

Catalog Number:

NBP1-97491-0.05mg

Hsp47 Antibody (M16.10A1) was used to determine the level of HSP47 after knockdown using siRNA in NIH3T3 mouse fibrobast cell line. The goal of the experiment was to select the best siRNA for use in proof of concept therapy for the treatment of fibrosis. This antibody was selected based reactivity to mouse protein, price and availability at the time of purchase.

Experimental Design and Results Summary

Applications

Western Blot

Sample

NIH3T3 whole cell lysate

Primary Incubation

1:1000 dilution, overnight incubation at 4 degrees in diluted Licor odyssey blocking buffer (1:1 with TBS) containing 0.1% tween-20

Blocking Agent

Licor Odyssey blocking buffer

Secondary Incubation

1:10,000 dilution of goat anti-mouse IR800CW antibody from Licor, 1.5 hour incubation at room temperature in diluted Licor odyssey blocking buffer (1:1 with TBS) containing 0.1% tween-20 and 0.02% SDS

Tertiary Incubation

N/A

Detection

Licor Odyssey IR scanner

Results Summary

Based on quantitation of the bands on the blot using ImageJ, siRNA #3 targeting the mouse HSP47 protein had the best knockdown effect (>70%). All protein levels were normalized against the level of GAPDH (loading control).

Additional Notes

None

Related Categories

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Summary

The Good

Worked right out of the box using our standard Western protocol and the manufacturer's recommended dilution of 1:1000.

The Bad

None

The Bottom Line

This antibody worked surprisingly well without the need for additional optimization. We were able to proceed with our experiments using results from this initial screening of siRNAs targeting HSP47.

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