Phospho Antibody With Great Sensitivity And Low Background

November 15, 2016

Neuroscience
Wright State University
Graduate Student

Overall

Quality of Results

Ease-of-Optimization

What do these ratings mean?
Write a Review

Company:

Cell Signaling Technology

Product Name:

Phospho-Akt (Ser473)

Catalog Number:

4060P

We used this antibody in our lab to look for activation of AKT in primary vascular smooth muscle cells. It worked without much optimization at a low concentration. We were able to reuse the buffer containing the antibody at least once. We highly recommend this antibody.

Experimental Design and Results Summary

Applications

Western Blot

Sample

Primary vascular smooth muscle cell protein extracts containing phosphatase inhibitors, 30-40ug/well during SDS page

Primary Incubation

1:1000 dilution, 24hrs. at 4C

Blocking Agent

5% BSA in TBST

Secondary Incubation

1:10,000, 1hr, at 25C

Tertiary Incubation

None

Detection

Chemiluminescence

Results Summary

Cell were lysed using lysis buffer containing phosphotase inhibitors (Roche) and protein concentrations were determined with BCA. Equal amounts of their protein extracts were separated by SDS-PAGE on 4-12% (w/v) polyacrylamide resolving gels and the proteins were transferred onto a PVDF membrane. The membrane was probed with AKT antibody (CST #4060P) , followed by its corresponding secondary antibody (anti-rabbit) conjugated to HRP. The membrane was developed using an ECL substrate and visualized using the ChemiDoc MP imaging system (Biorad). Equal protein load was ensured by reprobing same blot for total AKT protein (CST #4691).Westernbot analysis shows dose dependent increase in band intensity with the treatment of our componud of interest as well as a positive control - PDGF. Phosphorylation of AKT is significantly elevated with the addition of PDGF and our compound of interest.

DOI-PMID

N/A

Additional Notes

None

Related Categories

Image Gallery

Summary

The Good

Consistency, reproducibility, low background, great customer service

The Bad

Price

The Bottom Line

Worth the investment to try this antibody based on our experience. Try contacting the company directly, they have great customer service and have offered us significant discounts.

Share your experience with other scientists. Write a Review! »

Join the discussion