Anti-Glucose Transporter GLUT4 antibody [mAbcam48547] (ab48547) at 5 µg/ml + Partial tagged recombinant protein to GLUT4 at 0.1 µg

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

Performed under reducing conditions.

Predicted band size: 54 kDa
Observed band size: 30 kDa why is the actual band size different from the predicted?



ab48547 gave a positive signal against the partial recombinant GLUT4 protein which has an expected molecular weight of 30 kDa.

Overlay histogram showing NSO cells stained with ab48547 (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 (ab48547, 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 NSO cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.