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Our Abpromise guarantee covers the use of ab3765 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||1/100 - 1/500. Predicted molecular weight: 60 kDa.|
|IHC-P||1/50 - 1/100.|
|ICC/IF||Use a concentration of 10 µg/ml.|
|ChIP||Use 10µl for 106 cells.|
ab3765 stained HCT116 cells. The cells were 100% methanol fixed for 5 minutes at room temperature and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab3765 at 10µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was 150081 used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.
Western blot analysis using whole HL60 cell lysate: PRMT3 (Arrow) was detected using ab3765 (Lane B) but not pre-immune serum (lane A). Secondary HRP-anti-rabbit was used for signal visualization with chemiluminescence.
Sonicated Chromatin prepared from untreated or 17beta-estradiol (E2) treated MCF7 cells was subjected to the ChIP procedure with ab3765 to PRMT3 and the immunoprecipitated chromatin was analysed in the proximal region of the estrogen-responsive pS2 promoter (as shown above) and quantified by real-time PCR (values are % of inputs). The primers are designed to follow the nucleosome E (including the Estrogen Responsive Element ERE). 10