Loading Control Works Every Time

April 12, 2023

BlueskyReddit
IKP/RBCT
Robert Bosch Gesellschaft für medizinische Forschung mbH
Graduate Student

Overall

Quality of Results

Ease-of-Optimization

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Company:

Sigma-Aldrich (Merck)

Product Name:

Monoclonal Anti-β-Actin antibody

Catalog Number:

A5316

Clone Number:

AC-74

This mouse mAb against beta-Actin is our daily-use loading control for Western Blot detection. The concentration needed is very low, the solution is stable for a long time and the signals are always bright and clear and easy-to-detect.

Experimental Design and Results Summary

Applications

Western Blot

Sample

Whole cell lysates of human (cancer) cell lines blotted onto nitrocellulose membrane.

Primary Incubation

1:5000

Blocking Agent

5 % dry milk TBS-T

Secondary Incubation

1:2000

Tertiary Incubation

N/A

Detection

Chemiluminescence (ECL), ChemiDoc (Biorad)

Results Summary

In our hands, anti-beta-Actin from Sigma yields clear bands after a very short exposure (usually < 1 s) at approx. 42 kDa. We use it as our daily loading control for detection of other proteins.

DOI or PMID #

N/A

Additional Notes

Since the antibody is binding well and actin is usually quite abundant, stripping is not recommended. Better to detect it in the end in a sequential detection. Aliquot upon arrival. Ready-to-use antibody solution is stable at -20°C for several weeks and can be reused multiple times.

Image Gallery

Summary

The Good

Always works fine, remains stable.

The Bad

Stripping afterwards problematic.

The Bottom Line

This antibody yields high signals, is stable for a long time (even when already diluted) and is a convenient loading control for all of your Western Blots. Highly recommended.

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