Puma a/ß Primary Antibody (D-20) from Santa Cruz Biotechnology

March 04, 2013

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School of Cancer Studies
University of Birmingham
Research Technician / PhD Student
Example Western blot data generated using the Santa Cruz Puma a/ß D-20 primary antibody.

Company:

Santa Cruz Biotechnology

Product Name:

Puma a/ß Primary Antibody (D-20)

Catalog Number:

sc-20536

Image

In my assays, I was investigating the change in expression of Bcl-2 family member proteins in response to apoptotic stimuli in a range of cell lines using whole-cell lysate Western blotting under denaturing conditions. I particularly wanted a Puma antibody which detected both the alpha and beta isoforms of the protein as I was trying to corroborate published data on expression of the beta isoform specifically. Some other vendors of Puma antibodies were vague as to whether their product could pick up other isoforms, whereas this product was specifically designed to pick up both isoforms.

Experimental Design and Results Summary

Applications

Western blotting

Sample

Human B cell lymphoma cultured cell lines. Whole cell lysates in Urea-GSB (gel sample buffer) run on pre-poured 4-12% Bis-Tris Novex gels, 20 µg protein per well

Primary Incubation

Puma a/ß D-20, 1/200 in blocking agent, overnight at 4°C

Blocking Agent

5% non-fat milk in PBS-Tween, 1 hour at room temperature

Secondary Incubation

Donkey anti-Goat IgG HRP conjugate (sc-2020), 1/5000 in blocking agent, 1 hour at room temperature

Tertiary Incubation

N/A

Detection

ECL reagent (GE Healthcare RPN2106), developed using a Kodak X-OMAT 1000a automatic developer, captured on Amersham Hyperfilm ECL (28-9068-35)

Results Summary

Initially I had a lot of trouble using this antibody and struggled with very high background levels, however once I changed my secondary antibody to the one described above (I was previously using Sheep anti-Goat) this problem was much improved and the background problem was totally resolved. There are some non-specific bands but these do not run close to the correct bands (which ran at the expected size, which is ~26kDa for Puma alpha). The signal strength is good and I usually get nice results between 3-5 minutes of development time.

Additional Notes

I started using this antibody a few years ago and found that this was the best for detecting both the alpha and beta isoforms at that time, however many new monoclonal Puma antibodies have come on the market since then that are likely to give lower background and fewer non-specific bands. I have not tried any of the monoclonal antibodies but a colleague has and finds them to be very good; again the problem is whether you are interested in all isoforms or a specific one as I notice the new monoclonal from Santa Cruz is specific for only the alpha isoform.

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Example Western blot data generated using the Santa Cruz Puma a/ß D-20 primary antibody.

Summary

The Good

Gives consistent results in a short development time. Antibody keeps well at 4°C for at least 6 months.

The Bad

Must be used with a goat secondary antibody which has been raised in sheep and there are a few non-specific bands.

The Bottom Line

Not the best antibody I have used, but for my beta isoform studies, I have found it to be better than other available polyclonals. If you are interested in the alpha isoform alone I would try a monoclonal.

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