All lanes : Anti-SCD1 antibody (ab39969) at 1 µg/ml

Lane 1 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg
Lane 2 : HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg
Lane 3 : Wild-type HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 4 : Empty Lane
Lane 5 : SCD knockout HeLa whole cell lysate at 20 µg

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 41.5 kDa
Observed band size: 36 kDa why is the actual band size different from the predicted?
Additional bands at: 95 kDa. We are unsure as to the identity of these extra bands.

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% Milk before being incubated with ab39969 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to IRDye^® 800CW (green).

Loading control: alpha Tubulin ab7291 at 0.1 µg/mL visualised using a Goat anti-Mouse IgG H&L (IRDye^® 680RD, red).

All lanes : Anti-SCD1 antibody (ab39969) at 2 µg/ml

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : SCD knockout HeLa cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : HEK-293 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 41.5 kDa
Observed band size: 36 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab39969 observed at 36 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab39969 was shown to react with SCD1 in wild-type HeLa cells in western blot with loss of signal observed in SCD1 knockout cell line ab262177 (SCD1 knockout cell lysate ab257658). Wild-type and SCD1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab39969 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at 2 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

ICC/IF image of ab39969 stained HeLa cells. The cells were 4% formaldehyde (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 (ab39969, 10µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899 Dylight 488 goat anti-rabbit 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.

All lanes : Anti-SCD1 antibody (ab39969) at 2 µg/ml

Lane 1 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lane 2 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size: 41.5 kDa
Observed band size: 37 kDa why is the actual band size different from the predicted?