Highly Specific Phospho-S6 Antibody

December 10, 2015

Research
University of North Carolina at Chapel Hill
Laboratory Technician

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Company:

Cell Signaling Technology

Product Name:

Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb

Catalog Number:

#4858

Our lab investigates the role of kinases in several cancers, and we are often performing Western Blots. Our kinase work led us to probe the influence of kinase levels on specific proteins, including the S6 ribosomal protein. For purposes of our research, we needed to detect levels of phospho-S6 kinase in 786-0 cell lysates.We chose this CST antibody because of their reputable customer service and generally well-developed antibodies.

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

BioRad Clarity ECL with a Chemiluminescent Imager

Results Summary

Our lab was able to accurately detect levels of the phosphorylated ribosomal S6 protein in 786-0 cell lysates by using this specific antibody. This allowed us to draw further conclusions about the influence of the mTOR signaling cascade on protein translation in cancer cell lines. Using autoradiography film and a low-strength mixture of ECL, there was very little background and a strong signal. Around 15-30 seconds was sufficient to produce bands on film that were strong but not overpowering.

Additional Notes

Start out with a low-strength ECL mixture and move up as needed. The signal is generally quite strong.

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Summary

The Good

Strong signal, low background, and a reliable manufacturer

The Bad

Expensive

The Bottom Line

Our lab prefers Cell Signaling Antibodies because of their rigorous validation protocol for all of their antibodies, and their prompt customer service if there are any problems or questions. This specific antibody lived up to their typical standard of specificity with low background, and for that reason we will continue to use it to detect phosphorylated ribosomal S6 protein levels in cell lysates.

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