Anti-sLea (KM231) Antibody from EMD Millipore

April 30, 2013

Biochemistry and Molecular Biology
University of Nebraska Medical Center
Postdoctoral Research Associate

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Expression of sLea antigen in different cancer cells as determined by Western blotting.

Company:

EMD Millipore

Product Name:

Anti-sLea (KM231) Antibody

Catalog Number:

565942-100UG

Image

sLea is metastatic-promoting, selectin ligand predominantly occuring on the surface of cancer cells. This carbohydrate antigen is built on type-1 structures synthesized by the sequential actions of various glycosyltransferases. Typical structure of terminal tetrasaccharide contains the sequence of NeuAcα2,3Galβ1,3(Fucα1,4)GlcNAc attached to core-1 or core-2 structures. Elimination/reduction of this antigen on cancer cells greatly reduces the metastatic property of cancer cells.

Experimental Design and Results Summary

Applications

Western blotting and confocal microscopy were carried-out to assess the expression of this selectin ligand in different cancer cells.

Sample

Lysate from prostate, lung and colon cancer cells were used to perform Western blotting; whole cells were used for confocal studies.

Primary Incubation

Anti-sLea (KM231) antibody (cat #565942-100ug). For Western blotting, incubation time: overnight (15 hours) at 4°C, dilution 1:1000 (0.25 µg/ml) in Western blotting blocking agent. For confocal microscopy, incubation time: 1 hour on ice, dilution 1:100 (2.5 µg/ml) in confocal blocking agent.

Blocking Agent

For Western blotting: 5% non-fat milk prepared in TBST (137 mM Sodium Chloride, 20 mM Tris, 0.1% Tween-20, pH 7.5); blocking for 1 hour at room temperature. For confocal studies: 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), pH 7.2; blocked for 1 hour on ice.

Secondary Incubation

Western blotting: Donkey Anti-mouse IgG HRP (Jackson Immunoresearch Laboratories, cat # 715-035-151) diluted 1:10000 in Western blocking buffer. Incubation: 1 hour at room temperature. Confocal: Donkey Anti-mouse IgG coupled to Alexa 488 (Jackson Immunoresearch Laboratories, cat # 715-545-150) diluted 1:100 in confocal blocking buffer. Incubation: 1 hour at room temperature

Tertiary Incubation

N/A

Detection

For Western blotting: Pierce ECL Western Blotting Substrate (cat# 32106) on GeneMate autoradiography films (BioExpress, cat# F-9023-5X7). For confocal: Zeiss 510 Meta Confocal Laser Scanning Microscope

Results Summary

The antibody Anti-sLea (KM231) gives strong signal with low background. We used this antibody for immunoprecipitation and Proximity Ligation Assay (PLA) studies as well (Chachadi et al., PLoS One. 2013;8(2):e57416). SLea antibody could detect as low as 10 µg of total lysate protein in Western blotting.

Additional Notes

I would highly recommend this antibody to study sialyl lewis a antigen expressed on various proteins as well as on different mucins. The antibody is highly specific in detecting the carbohydrate antigen.

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Expression of sLea antigen in different cancer cells as determined by Western blotting.

Summary

The Good

This antibody is highly specific and can detect as low as 10 µg of whole cell lysate during Western blotting. The background level is very low.

The Bad

None, but price may be little expensive

The Bottom Line

The antibody is a really great tool to study sialyl lewis a carbohydrate epitope.

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