Immunohistochemistry analysis staining Rhodopsin in mouse retina with ab98887 at 1/1000. Secondary Antibody: FITC Goat Anti-Mouse (green). Counterstain: DAPI (blue) nuclear stain.

OS - photoreceptor outer segment; IS – inner segment; ONL – outer nuclear layer; OPL – outer plexiform layer; INL – inner nuclear layer; IPL – inner plexiform layer; GCL – ganglion cell layer.

All lanes : Anti-Rhodopsin antibody [Rho 4D2] (ab98887) at 1/1000 dilution

Lane 1 : Bovine photoreceptor membrane tissue lysate at 10 µg
Lane 2 : Bovine photoreceptor membrane tissue lysate at 5 µg
Lane 3 : Bovine photoreceptor membrane tissue lysate at 2.5 µg

Predicted band size: 38 kDa
Observed band size: 110,35,75 kDa why is the actual band size different from the predicted?

Anti-Rhodopsin antibody [Rho 4D2] (ab98887) at 1/1000 dilution (2.5 hours at room temperature with shaking) + A549 (human lung carcinoma cell line) whole cell lysate at 15 µg

Secondary
Goat Anti-Mouse IgG (HRP) for 1 hour at room temperature with shaking at 1/1000 dilution

Predicted band size: 38 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

Band appears at ~75 kDa indicating detection of the Rhodopsin dimer.

 

Block: 5% Skim Milk Powder in TBST.

 

Color Development: Chemiluminescent for HRP for 5 min at room temperature.

Overlay histogram showing Y79 (Human retinoblastoma cell line) cells stained with ab98887 (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 (ab98887, 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 IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Y79 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.