CD15s (CSLEX)-Anti-Sialyl Lewis x Antibody from BD Biosciences

April 08, 2013

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Biochemistry and Molecular Biology
University of Nebraska Medical Center
Postdoctoral Research Associate
Flow cytometric analysis of sLex expression on HCT15 cells. [Please refer the published article and fig 6. Chachadi et al, Int J Biochem Cell Biol 43: 586-593 (2011) for detailed results]

Company:

BD Biosciences

Product Name:

CD15s (CSLEX)-Anti-sialyl lewis x antibody

Catalog Number:

551344

Image

The aim of the study was to check the epigenetic regulation of the synthesis of selectin ligands (sialyl lewis x and sialyl lewis a). Upon searching the literature, many researchers used this antibody to recognize sLex carbohydrate antigen by flow cytometry. We wanted to investigate whether the expression of this selectin ligand (sLex ) is under the control of epigenetics through glycosyltransferases involved in the synthesis of this antigen.

Experimental Design and Results Summary

Applications

We performed Western blotting, flow cytometry and confocal microscopy to determine the expression levels of this antigen before and after various drugs known to be involved in epigenetic studies.

Sample

We used colon cancer cells for flow cytometry and confocal studies. Cell lysate from the same samples were used for Western blotting.

Primary Incubation

CD15s (CSLEX)-Anti-sialyl lewis x antibody, cat #551344. For Western blotting: Incubation time: overnight (15 hour) at 4°C, diluted 1:1000 (0.5 µg/ml) in Western blocking agent. For flow cytometry and confocal microscopy: Incubation time: 1 hour on ice, diluted 1:200 (2.5 µg/ml) in flow cytometry and confocal blocking agent.

Blocking Agent

Blocking Agent for Western blotting: 5% Non-fat milk prepared in tris-buffered saline with tween (TBST: 137 mM Sodium Chloride, 20 mM Tris, 0.1% Tween-20, pH 7.5). Blocking time: 1 hour at room temperature. Blocking agent for flow cytometry and confocal: 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), blocked for for 1 hour on ice.

Secondary Incubation

Western blotting: Donkey Anti-mouse IgM HRP (Jackson Immunoresearch Laboratories, Cat # 715-035-140), diluted 1:10,000 in blocking buffer; incubation: 1 hour at room temperature. Flow cytometry and confocal: Goat Anti-mouse IgM coupled to DyLight 488 and 594 (Jackson Immunoresearch Laboratories, Cat # 115-485-075); for flow cytometry: 1:200 dilution in 3% BSA prepared in PBS, For confocal: 1:100 in 3% BSA prepared in PBS.

Tertiary Incubation

N/A

Detection

For Western blotting: Pierce ECL Western Blotting Substrate Cat#32106 on GeneMate autoradiography films, Cat#F-9023-5X7. For flow cytometry: FACS Vantage equipped with Cell Quest-Pro software from Becton Dickson. Confocal: Zeiss 510 Meta Confocal Laser Scanning Microscope

Results Summary

The antibody CD15s (Cat#551344) against carbohydrate antigen sialyl lewis x, gives very good signal strength with low back ground on Western blots. Usually carbohydrate antibodies tend to give lot of background especially if they are IgM isotype. This antibody also gave very good signals in flow cytometry and confocal with very low background. We used this antibody for immunoprecipitation studies also. (Chachadi et al, Int J Biochem Cell Biol 43: 586-593 (2011).) We used three different lots and the results were consistent.

Additional Notes

I would highly recommend this antibody to study sialyl lewis x antigen. The antibody is highly specific in detecting the carbohydrate antigen despite being IgM isotype. There are very good secondary antibodies from different companies to detect this IgM isotype. We used Agarose-L from Pierce (cat #20520) to isolate immunocomplexes while using this antibody for IP studies.

Image Gallery

Flow cytometric analysis of sLex expression on HCT15 cells. [Please refer the published article and fig 6. Chachadi et al, Int J Biochem Cell Biol 43: 586-593 (2011) for detailed results]

Summary

The Good

This antibody is highly specific and can detect as low as 25 ug of whole cell lysate during Western blotting. The background level is very low. Another great thing about this antibody is that it can be stored at 4°C.

The Bad

I would recommend changing the vial and its cap; you cannot see how much is left unless you pipette out.

The Bottom Line

The antibody is a really great tool to study sialyl lewis x which is an important selectin ligand both in lymphocyte trafficking and hematogenous metastasis of cancer cells. Ratings

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