Anti-CRMP2 抗体 [1B1] (ab62539)

All lanes : Anti-CRMP2 antibody [1B1] (ab62539) at 1/1000 dilution

Lane 1 : Wild-type A549 cell lysate at 20 µg
Lane 2 : DPYSL2 knockout A549 cell lysate at 20 µg
Lane 3 : HeLa cell lysate at 20 µg
Lane 4 : A549 Nuclear Fraction cell lysate at 20 µg
Lane 5 : Recombinant Human CRMP2 protein (ab114269) cell lysate at 0.2 µg

Performed under reducing conditions.

Predicted band size: 65 kDa
Observed band size: 63 kDa why is the actual band size different from the predicted?

Western blot: Anti-DPYSL2 antibody [1B1] (ab62539) staining at 1/1000 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab62539 was shown to bind specifically to DPYSL2. A band was observed at 63 kDa in wild-type A549 cell lysates with no signal observed at this size in DPYSL2 knockout cell line. To generate this image, wild-type and DPYSL2 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

Anti-CRMP2 antibody [1B1] (ab62539) at 1/2500 dilution + CRMP2 antibody [1B1] (ab62539) at 1/2500 dilution + SK N BE (Human neuroblastoma) Whole Cell Lysate at 10 µg

Secondary
Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 65 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?

Overlay histogram showing SH-SY5Y cells stained with ab62539 (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 (ab62539, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.