Great Phospho-Src Family Antibody

December 18, 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-Src Family (Tyr416) (D49G4) Rabbit mAb

Catalog Number:

#6943

Our lab works to elucidate the impact of kinases in cancer progression, and our research often includes Western Blots in order to determine kinase protein and activity levels in various cancer cell lines. In particular, we were working to detect endogenous levels of the phosphorylated Src family of kinases in 786-0 cell lysates. We chose this CST antibody because of their reputable customer service and 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

ECL --> Autoradiography Film via HRP-Conjugate Secondary Antibody

Results Summary

Using this antibody, we were able to detect accurately the levels of phosphorylated Src family kinases in 786-0 cell lysates. This allowed us to draw further conclusions about the influence of the Src family of kinases upon cancer progression in several types of cancers. Using autoradiography film and a low-strength mixture of ECL, there was very little background and a strong signal. Around 15-30 seconds of exposure time was sufficient to produce adequate bands on film.

Additional Notes

Is non-specific within the Src family of kinases

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Summary

The Good

Strong signal, low background, and a reliable manufacturer

The Bad

Expensive

The Bottom Line

This antibody has proven to be a good investment for our lab. Very little optimization was needed, and the almost complete lack of background signal is superb. While the price tag is expensive, not having to optimize the experiment, try multiple antibodies, or fumble around with bad customer service is well worth it. I highly recommend this particular antibody for the detection of phosphorylated Src family kinases.

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