All lanes : Anti-PAX5 antibody [mAbcam52588] (ab52588) at 1 µg/ml

Lane 1 : Raji (Human Burkitt's lymphoma cell line) Whole Cell Lysate (blocked with 1% milk)
Lane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate (blocked with 1% Milk)

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 42 kDa
Observed band size: 45 kDa why is the actual band size different from the predicted?
Additional bands at: 50 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 20 minutes

Overlay histogram showing Ramos cells stained with ab52588 (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 (ab52588, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse 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 Ramos cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.