Antibody Did Not Work as Expected for the Western Blots

July 03, 2020

Medicine
Institute of Virology, TU Munich
Graduate Student

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Company:

R&D Systems

Product Name:

Mouse Galectin-9 Antibody

Catalog Number:

AF3535

Clone Number:

N/A

Mouse alveolar macrophage cell line was tested for expression of Galectin-9 (Gal-9) after siRNA mediated knockdown. The antibody did not work as claimed, even at higher concentrations for western blots.

Experimental Design and Results Summary

Applications

Western Blot

Sample

MH-S (Mouse Alveolar Macrophage cell line)

Primary Incubation

1:1000, 1:500, 1:200

Blocking Agent

5% milk

Secondary Incubation

1:5000, 1:10,000 Rabbit anti-goat secondary antibody

Tertiary Incubation

Approximately 3 minutes with ECL blot developing solution

Detection

Blot development using fully functional apparatus, scans from as early as 30s till 15 minutes

Results Summary

MH-S cells were treated with siRNAs against Galectin-9 and compared against Negative control siRNA treated cells and mock-transfected cells. Total soluble proteins (TSP) from all the experimental groups were extracted using RIPA buffer supplied with protease inhibitor by standard protocol. TSP were quantified by the Bradford method and equal amounts of TSP were subjected to Western blot analysis to determine knockdown efficiency of candidate siRNAs.More than 10 bands were observed after a long exposure time of 15 minutes, rather than a specific expected band. It was even hard to determine the specific band of Gal-9, making the results impossible to interpret. Please check the image provided. The image is totally unprocessed and/or edited.

DOI or PMID #

N/A

Additional Notes

Samples were run on 12.5% agarose gel and 0.45u membrane was used for transfer

Image Gallery

Summary

The Good

Delivery

The Bad

Did not work as claimed, poor quality of results in Macrophage cell line

The Bottom Line

I would not recommend this product for WB analysis from macrophage cell line. It did not work with cell lysate and cell supernatant. In general, for analysis of Gal-9 expression levels, please opt for a flow cytometry-based methodology.

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