Good Antibody to Detect Human TGF Beta 1 Under Non-Reducing Conditions

September 06, 2018

The Cell and Molecular Biology
The Scripps Research Institute
Assistant Professor

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Company:

Bxcell ( Bio X Cell )

Product Name:

InVivoMAb anti-TGF-β1 (clone 1D11.16.8)

Catalog Number:

BE0057

TGF-β1 is an important growth factor ubiquitously expressed in normal and cancer cells. It is produced as a Small Latent Complex (SLC), containing the mature TGF-β1 homodimer, which should be released from the SLC to become active and capable of binding to the TGF-β1 receptors. We analyzed the binding of select LTBPs (Latent TGF-β Binding Proteins) in the binding of the TGF-β1 SLC, resulting in the formation of a Large Latent Complex (LLC). To investigate the functional importance of LTBPs in the secretion and presentation of TGF-β1 to the cells, we needed to identify the TGF-β1 protein secreted by human tumor cells. The mouse anti-TGF-β1 monoclonal antibody (mAb) 1D11.16.8 from BioXCell (Bxcell) was used to analyze the secreted TGF-β1 in western blot assays under reducing and non-reducing conditions. The 1D11.16.8 mAb nicely recognized TGF-β1 as a mature homodimer and as SLC in the concentrated conditioned medium from human cells expressing LTBP-3 and TGF-β1.

Experimental Design and Results Summary

Applications

Western Blot

Sample

Concentrated conditioned medium from human cells producing TGF-β1

Primary Incubation

Antibody at a final concentration of 10 microgram/ml, ON incubation with gentle rocking in a cold room

Blocking Agent

5% milk in PBS-0.05% Tween 20

Secondary Incubation

Stabilized goat anti mouse IgG HRP conjugate, 1:5,000 in blocking buffer

Tertiary Incubation

N/A

Detection

West Pico

Results Summary

The mAb 1D11.16.8 from BioXCell nicely recognized TGF-β1 in the samples of conditioned medium (CM) from the cells transfected with TGF-β1. The antibody appears highly specific since no protein bands were visualized in the CM from the cells that were transfected with the empty vector. The antibody appears to work only under non-reducing conditions.

DOI or PMID #

N/A

Additional Notes

The image demonstrates 2 protein bands, which would correspond to the mature TGF-β1 (expected mol. wt. ~24 kDa) and the SLC (expected mol wt ~95 kDa). The antibody appears to work only under non-reducing (-DTT) conditions.

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Summary

The Good

The antibody appears to be highly specific as no non-specific protein bands were detected even after prolonged exposure.

The Bad

In our hands, the reliable detection of TGF-β1 required the use of concentrated conditioned medium. We also used a relatively high concentration of this mAb (10 microgram/mL instead of usual 1-2 microgram/mL) to increase the efficiency of TGF-β1 detection.

The Bottom Line

The mouse mAb 1D11.16.8 from BioXCell (bxcell) appears to be a highly specific antibody detecting TGF-β1 protein by western blotting under non-reducing conditions.

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