Reliable and Consistent Beta Actin Loading Control

June 03, 2026

BlueskyReddit
Bioengineering
SII
Principal Investigator

Overall

Quality of Results

Ease-of-Optimization

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Company:

Cell Signalling Technology CST

Product Name:

Anti-Beta-Actin Antibody

Catalog Number:

4967S

Clone Number:

13E5

We used the CST Anti-Beta-Actin (13E5) rabbit monoclonal antibody as a loading control in Western blot experiments conducted on multiple cell lines, including HeLa, HEK293, and primary mouse neurons. This antibody consistently yielded a sharp, clean band at approximately 42 kDa, with minimal background noise. This makes it an excellent and reliable reference for protein normalization in our studies.

Experimental Design and Results Summary

Applications

Western Blot

Sample

Human HeLa cells and HEK293 cells; also validated in primary mouse cortical neurons

Primary Incubation

1:1000 dilution in 5% BSA/TBST; overnight at 4°C with gentle rocking

Blocking Agent

5% non-fat dry milk in TBST for 1 hour at room temperature

Secondary Incubation

Anti-rabbit HRP conjugated secondary antibody (1:2000), 1 hour at room temperature

Tertiary Incubation

NA/ Standard Western Blot Used

Detection

Enhanced chemiluminescence (ECL) using SuperSignal West Pico PLUS.

Results Summary

The Anti-Beta-Actin (13E5) antibody reliably detected a single clean band at ~42 kDa across all tested cell lines with no non-specific bands. Signal intensity was consistent across experiments, making it an ideal loading control for normalization. Background was minimal even without extended optimization, and the antibody performed well after up to 5 freeze-thaw cycles.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Extremely consistent and reproducible signal with no optimization needed. Works right out of the box at 1:1000 dilution. Single clean band with virtually no background. Excellent lot-to-lot consistency across multiple orders.

The Bad

Price point is higher than competitors. Rabbit monoclonal format means it cannot be used alongside other rabbit primaries in multiplex experiments without a special secondary strategy.

The Bottom Line

The gold standard for beta-actin detection, worth every penny for labs that require reliable, publication quality loading controls..

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