A Good Antibody for Detecting Human NCR1 (Nkp46, CD335)

June 23, 2016

Division of BioMedical Sciences, Faculty of Medicine
Memorial University of Newfoundland
Research Assistant

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Company:

abcam

Product Name:

Anti-NCR1 antibody [MM0491-8F24] (ab89877)

Catalog Number:

ab89877

NCR1 (also known as Nkp46 or CD335) is a useful marker for identifying NK cells in the peripheral blood. Our lab routinely performs FACS staining of human peripheral blood mononuclear cells to identify lymphocyte subsets to study their biology. the particular goal of this experiment was to identify circulating NK cells in individuals with different disease states. This antibody was chosen because (a) it recognizes an extracellular epitope of NCR1, thus fixing and permeabilization are not required for FACS staining, and (b) it has been previously validated for flow cytometry by the manufacturer.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Human PBMC isolated by Ficoll density gradient separation

Primary Incubation

Anti-human NCR1 antibody (ab89877), incubated with approximately 1x10^6 cells for 1 hour on ice at 1:50 (1.0ug) dilution in a total volume of 100ul PBS+2% FCS, 1mM EDTA, 0.1% sodium azide.

Blocking Agent

N/A

Secondary Incubation

Alexa Fluor 647-conjugated donkey anti-mouse IgG (H+L) (Jackson 715 605 151), incubated for 1 hour on ice in the dark at 1:800 dilution in a total volume of 100ul PBS+2% FCS, 1mM EDTA, 0.1% sodium azide.

Tertiary Incubation

N/A

Detection

FACSCalibur (Becton Dickinson)

Results Summary

As shown in the FACS histogram, approximately 13% of lymphocytes were identified as expressing NCR1 (open histogram), which falls within the expected range for NK cells. NCR1+ cells were variable in their expression. Staining could have been brighter and more uniform. Background staining was minimal given that NCR1- cells resembled isotype control cells (shaded histogram). A negative control should be included in your experiment when using this antibody since there is not a large degree of separation between NCR1+ and NCR1- cells.

Additional Notes

Gating was performed on the lymphocyte population for FACS analysis

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Summary

The Good

Verified for flow cytometry with recommended dilutions - worked well right out of the box with little optimization.

The Bad

Staining could be brighter - you might want to optimize your secondary to achieve this. Only available in one size at the moment (no test size available). Would be nice if it came directly-labelled for flow cytometry.

The Bottom Line

Recommended for identification of NCR1-expressing NK cells by flow cytometry. You may need to perform some optimization to achieve your desired staining brightness.

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