Mouse mAb Against Human Neutrophil Elastase

September 12, 2017

The Cell and Molecular Biology
The Scripps Research Institute
Assistant Professor

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Company:

Santa Cruz Biotechnology, Inc.

Product Name:

Neutrophil Elastase Antibody (D-7)

Catalog Number:

sc-365950

Detection of neutrophil elastase (NE) is important in the studies of inflammation, specifically in studying the inflammatory responses of neutrophils. NE is a powerful serine protease that is expressed mainly by neutrophils, which release NE upon stimulation. Specific NE activity is measured by a peptide cleavage assay, but it is also important to know how much of total NE has been released by neutrophils under specific conditions. Total NE could be determined by western blotting in comparison with the known quantities of purified NE. The mouse monoclonal antibody sc-365950 (D-7) from Santa Cruz was designed to detect human NE by western blotting and other techniques, including IP, IF and ELISA. We tested this antibody for the ability to detect human purified NE in western blotting.

Experimental Design and Results Summary

Applications

Western Blot

Sample

Purified neutrophil elastase, 0.5 microgram; U937 cell lysate, 50 micrograms; granule contents of 1x10^6 human neutrophils

Primary Incubation

2 ug/ml in PBS supplemented with 5% nonfat milk and 0.05% Tween 20; overnight, cold room

Blocking Agent

5% nonfat milk in PBS/0.05% Tween 20

Secondary Incubation

Goat anti-mouse IgG conjugated with HRP; 1:3,000; 1 hr, room temperature

Tertiary Incubation

None

Detection

West Pico

Results Summary

In our hands, the antibody sc-365950 did not recognize either purified NE (lane 1) or NE within neutrophil granule content (lane 3). This antibody did produce a positive band of approximately 30 kDa in the U-937 cell lysate (lane 2), which is interpreted by the manufacturing company as NE-specific band. However, U-937 cells originate from a human histiocytic lymphoma and these cells should not produce huge quantities of NE (as indicated by the company's tests).

DOI or PMID #

N/A

Additional Notes

The lack of NE in U-937 cell lysate could be confirmed with the NE-specific rabbit antisera from Athens.

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Summary

The Good

None detected

The Bad

This antibody did not recognize human NE from a natural source, i.e. neutrophils.

The Bottom Line

The mAb D-7 that was raised against a peptide sequence shared by human NE, but unfortunately this antibody does not recognize human NE, either purified from human neutrophils or within neutrophil granule content.

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