Western blot of Human 2831 in T47D cell lysate using ab2831.

ICC/IF image of ab2831 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab2831, 5µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

ab2831 can be used in ChIP. You can expect recruitment of AIB1on the pS2 promoter at some point during transcriptional activation. The amount of DNA precipiated is significant.

Sonicated Chromatin prepared from untreated or 17beta-estradiol (E2) treated MCF7 cells was subjected to the ChIP procedure with ab2831 to AIB1 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). 3 µl of ab2831 and 2x106 cells were used in each ChIP experiment. 

IHC image of ab2831 staining in human melanoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab2831, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.