Proximity Ligation Analysis with anti-cMyc pS62 and CIP2A antibodies, association of cMyc pS62 with CIP2A (red) in nuclei (DAPI, blue) of HeLa cells by ICC/IF. 

Ab78318 staining (green) and DAPH staining (blue) of c-Myc (phospho S62) in HeLa cells by ICC/IF.

 

All lanes : Anti-c-Myc (phospho S62) antibody [33A12E10] (ab78318) at 1 µg/ml

Lane 1 : Crude cell extracts of AGS (gastric adenocarcinoma) cells with Scr (scrambled) siRNA introduced into the cells as a negative control
Lane 2 : Crude cell extracts of AGS (gastric adenocarcinoma) cells transfected with a negative control siRNA
Lane 3 : Crude cell extracts of AGS (gastric adenocarcinoma) cells transfected with siRNA for c-Myc

Predicted band size: 49 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

IHC image of ab78318 staining in human normal cervical carcinoma 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 ab78318, 1µg/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.

Overlay histogram showing HL60 cells stained with ab78318 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab78318, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was a goat anti-mouse DyLight® 488 (IgG; H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HL60 cells fixed in 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.