Mouse mAb Against Human Neutrophil Elastase

September 08, 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 (NP57)

Catalog Number:

sc-53388

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-53388 (F-1) from Santa Cruz was designed to detect human NE by western blotting and other techniques, including IP, IF, IHC 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-53388 did not recognize either purified NE (lane 1) or NE within neutrophil granule content (lane 3). This antibody also did not produce any positive bands in the U-937 cell lysate that surprisingly is used as a positive control for NE by the manufacturing company (lane 2).

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

None

The Bad

This antibody did not recognize human NE

The Bottom Line

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

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