Great pSTAT1 Antibody

December 17, 2015

Research
University of North Carolina at Chapel Hill
Laboratory Technician

Overall

Quality of Results

Ease-of-Optimization

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Company:

Cell Signaling Technology

Product Name:

Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb

Catalog Number:

#9167

Our lab aims to elucidate the role of kinases in several cancers, and we are often performing Western Blots to determine kinase protein and activity levels in various cell lines. For purposes of our research, we needed to detect endogenous levels of STAT1 kinase phosphorylated at the Tyr701 residue in 786-0 cell lysates. We chose this antibody because of the reputable customer service and well-developed validation process for antibodies at Cell Signaling Technology.

Experimental Design and Results Summary

Applications

Western Blot

Sample

786-0 Cell Lysates

Primary Incubation

1:1000 Dilution Overnight in 5% BSA in 1X TBS-T at 4C

Blocking Agent

5% Milk in 1X TBS-T

Secondary Incubation

1:5000 dilution of HRP-conjugated Rabbit secondary in 5% Milk in TBS-T for 1 hour at RT

Tertiary Incubation

NA

Detection

ECL/FEMTO --> Autoradiography Film via HRP-Conjugated Rabbit Secondary Antibody

Results Summary

Using autoradiography film and a 1:1 ratio of ECL and FEMTO, there was minimal background and a strong signal. Around 15-30 seconds was sufficient to produce bands on film that were strong but not overpowering. Our lab was able to accurately detect levels of the phosphorylated STAT1 kinase in 786-0 cell lysates and differentiate between the individual alpha and beta isoforms in our control samples. This allowed us to draw further conclusions about the influence of various inhibitors on the STAT1 signaling cascade in cancer cell lines.

Additional Notes

Needs more than just ECL to come up on film.

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Summary

The Good

Highly specific, strong signal, well-validated

The Bad

Expensive

The Bottom Line

In particular, the ability to distinguish between multiple isoforms of the STAT1 kinase is impressive, and worth the price. I am yet to find a better antibody to detect the phosphorylated Tyrosine 701 residue of STAT1, and will continue to use this antibody for years to come. Highly recommended, and well worth it.

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