All lanes : Anti-Sortilin/NT3 antibody (ab16640) at 1 µg/ml

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : SORT1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 95 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab16640 observed at 100 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab16640 was shown to react with Sortilin/NT3 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264772 (knockout cell lysate ab257696) was used. Wild-type HeLa and SORT1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab16640 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 µg/ml and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Sortilin/NT3 knockout HAP1 cell lysate (20 µg)
Lane 3: NIH/3T3 cell lysate (20 µg)
Lane 4: 293T cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab16440 observed at 100 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab16640 was shown to specifically react with Sortilin/NT3 in wild-type HAP1 cells. No band was observed when Sortilin/NT3 knockout samples were examined. Wild-type and Sortilin/NT3 knockout samples were subjected to SDS-PAGE. ab16640 and ab8245 (loading control to GAPDH) were diluted 1 µg/mL and 1/10,000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

Lane 1 : Marker
Lanes 2-4 : Anti-Sortilin/NT3 antibody (ab16640) at 1 µg/ml

Lane 2 : Human Brain tissue lysate at 20 µg
Lane 3 : Mouse Brain tissue lysate at 20 µg
Lane 4 : Rat Brain whole cell lysate at 20 µg

Secondary
Lanes 2-4 : Goat polyclonal to Rabbit IgG H&L (HRP) Pre-Adsorbed at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 95 kDa
Additional bands at: 31 kDa (possible cleavage fragment)

Immunofluorescent staining for Sortilin/NT3 in rat cortical neurons using ab16640. The staining is cytoplasmic and punctate of many cells such as cortical neurons. The image was taken with a X20 objective.

Protocol details: Rats were intracardially perfused with 4% paraformaldehyde. Whole brain tissue was post-fixed overnight in the same fixative, cryoprotected in 20% sucrose and frozen in OCT and then cut on cryostat (30µm coronal sections). IHC was perfored in free floating with fixed tissues (rat brain sections). Primary antibody was incubated overnight at 1/3000 at room temperature