Okay Antibody to Detect Actin for Loading

May 02, 2019

Oncology
Johns Hopkins School of Medicine
Graduate Student

Overall

Quality of Results

Ease-of-Optimization

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Company:

Cell Signaling Technology (CST)

Product Name:

β-Actin (D6A8) Rabbit mAb

Catalog Number:

8457

Clone Number:

Our lab studies the role of mTOR signaling in T cell such as mTORC1 and mTORC2 signaling during T cell differentiation and function. With immunoblot analysis, we assess mTOR activity in WT T cells upon T cell activation. To normalize total protein content in certain experiments, we use actin as a control in our blots. This is an okay antibody with great specificity but short half life.

Experimental Design and Results Summary

Applications

Western Blot

Sample

Murine T cells

Primary Incubation

1:10000 in 4% BSA/TBST 4 deg overnight with shaking

Blocking Agent

5% milk/TBST

Secondary Incubation

1:5000 anti-Rabbit IgG HRP (CST) in 5% dry milk/TBST

Tertiary Incubation

None

Detection

UVP Imaging System

Results Summary

We are able to detect total beta-actin levels in T cell lysates. The signal is quite strong as a loading control.

DOI or PMID #

N/A

Additional Notes

Do not reuse antibody more then three to four times since you get unreliable results.

Image Gallery

Summary

The Good

Strong signal if used relatively fresh aliquot.

The Bad

Can not reuse many times compared to other CST antibodies.

The Bottom Line

Nice antibody to detect beta-actin as a loading control with specificity however not reliable after reusing it.

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