All lanes : Anti-BPR antibody [EPR5201] (ab108346) at 1/1000 dilution

Lane 1 : U87-MG cell lysate
Lane 2 : HT-1376 cell lysate
Lane 3 : MCF7 cell lysate
Lane 4 : PC-3 cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 37 kDa
Observed band size: 32 kDa why is the actual band size different from the predicted?

ICC/IF image of ab108346 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab108346, 1/100) overnight at +4°C. The secondary antibody (green) was ab96899, a goat anti-rabbit DyLight® 488 (IgG; H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM

Overlay histogram showing HeLa cells stained with ab108346 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab108346, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was a goat anti-rabbit Alexa Fluor® 488 (IgG; H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Immunohistochemical analysis of BPR in paraffin embedded Human breast carcinoma tissue, using ab108346 at a dilution of 1/500.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.