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Synthetic phosphopeptide derived from the region of human cFos that contains threonine 325.
Our Abpromise guarantee covers the use of ab27793 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||Use a concentration of 2 - 3 µg/ml.|
|ChIP||Use 1-3µg for 106 cells.|
|WB||1/1000. Predicted molecular weight: 41 kDa.|
|IP||Use at an assay dependent concentration. PubMed: 20410304|
Immunofluorescence analysis of c-Fos (phospho T325) was done on 70% confluent log phase HeLa cell treated with 200 nM of PMA for 20 minutes. The cell were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Phosc-Fos (phospho T325)Rabbit Polyclonal Antibody (ab27793) at 2 ug/ml in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate at a dilution of 1/2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with DAPI. F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin. Panel d is a merged image showing nuclear and cytoplasmic localization. Panel e is untreated cell with no signal. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
Chromatin Immunoprecipitation (ChIP) was performed using Anti-c-Fos (phospho T325) antibody (ab27793) 3 ug on sheared chromatin from 2 million A431 cells treated with EGF (200ng/ml), for 30 minutes. Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by 7500 Fast qPCR system with optimized PCR primer pairs for the promoter of active IL-6, CDKN1A gene, used as positive control target, and the SAT2, used as negative control target. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.