Anti-ESET IF (aka SETDB1)

June 11, 2014

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Biological Science
Florida State University
Assistant Professor

Company:

Cell Signaling Technology

Product Name:

Anti-ESET (C1C12) Rabbit mAb

Catalog Number:

2196

The purpose of this experiment was to identify an antibody that was suitable for detection of SETDB1 in human cells by indirect immunofluorescence.

Experimental Design and Results Summary

Applications

Immunofluorescence

Sample

Human hTERT-RPE1 cells

Primary Incubation

1:100 at room temperature for 60 minutes. Staining was performed in 1 x PBS and 0.1% Tween 20

Blocking Agent

Slide was pre blocked with 3% BSA in 1 x PBS and 0.1% Tween 20

Secondary Incubation

1:100 dilution of Alexa-488 Goat anti-rabbit secondary diluted in 1 x PBS and 0.1% Tween-20 for 60 minutes at room temperature

Tertiary Incubation

No tertiary incubation was performed

Detection

Direct labeled goat anti-rabbit antibodies

Results Summary

This antibody stains relatively well and detects the anticipated speckled appearance of SETDB1/ESET in the nucleus. The signal is relatively weak, but the background is low.

Additional Notes

Cells were grown directly on slides and washed briefly with 1 x PBS before fixing and extracting for 10 minutes at room temperature in 4% formaldehyde, 1 x PBS and 0.1% Triton-X100

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Summary

The Good

Works well by immunofluorescence despite being a weak signal

The Bad

No drawbacks were evident with this antibody

The Bottom Line

This antibody is suitable for detecting ESET/SETDB1 by immunofluorescence, but the signal is weak and may benefit from 1:50 as opposed to 1:100.

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