Anti-Mouse IgG, HRP-Linked Antibody Works Well as Secondary Antibody for Western Blotting

June 22, 2020

Radiation Oncology
UAB
Research Scientist

Overall

Quality of Results

Ease-of-Optimization

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Company:

Cell Signaling Technology

Product Name:

Anti-mouse IgG, HRP-linked Antibody

Catalog Number:

7076

Protein lysates from human breast cancer cells were electrophoresed and transferred to PVDF prior to probing for B-Actin. The Anti-mouse IgG, HRP-linked Antibody was used as secondary antibody for B-actin to determine loading efficacy.

Experimental Design and Results Summary

Applications

Western Blot

Sample

Human breast cancer cells

Primary Incubation

1:50,000 for 30 minutes at RT

Blocking Agent

5% BSA in TBS-T

Secondary Incubation

1:1000 for 30 minutes at RT

Tertiary Incubation

2 minutes with ECL reagents at RT

Detection

ECL Reagents

Results Summary

Loading appeared to even across all lanes. Another band was detected just below the B-actin band so 5% milk may be a better choice for blocking and antibody dilution. Only one second exposure was sufficient to detect sufficient B-actin levels.

DOI or PMID #

N/A

Additional Notes

N/A

Image Gallery

Summary

The Good

Very easy to use

The Bad

May not be used for 5% BSA but for milk instead

The Bottom Line

The secondary antibody appears to be quite specific for B-actin in western blotting. Since B-actin detection is usually quite sensitive, the duration of secondary antibody incubation and its concentration can be scaled down to prevent oversaturation of the protein band.

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