All lanes : Anti-Sortilin/NT3 antibody [EPR15010] (ab188586) at 1/1000 dilution

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: 92 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 - ab188586 observed at 100 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab188586 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. ab188586 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 1000 dilution 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.

Immunohistochemical analysis of paraffin-embedded Human brain tissue labeling Sortilin/NT3 with ab188586 at 1/250 dilution followed by prediluted HRP Polymer for Rabbit IgG. Counter stained with Hematoxylin.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Sortilin/NT3 knockout HAP1 cell lysate (20 µg)
Lane 3: SW480 cell lysate (20 µg)
Lane 4: HeLa cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab188586 observed at 92 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab188586 was shown to  react with Sortilin/NT3 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when Sortilin/NT3 knockout samples were examined. Wild-type and Sortilin/NT3 knockout samples were subjected to SDS-PAGE. ab188586 and ab8245 (loading control to GAPDH) were both diluted 1/2000 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.

All lanes : Anti-Sortilin/NT3 antibody [EPR15010] (ab188586) at 1/1000 dilution

Lane 1 : SH-SY5Y whole cell lysate
Lane 2 : Human hippocampus tissue lysate
Lane 3 : Human fetal brain tissue lysate
Lane 4 : Mouse brain tissue lysate
Lane 5 : 3T3-L1 whole cell lysate
Lane 6 : Rat brain tissue lysate

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 92 kDa
Observed band size: 92 kDa


Exposure time: 30 seconds

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Sortilin/NT3 antibody [EPR15010] (ab188586) at 1/2000 dilution

Lane 1 : SW480 cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : SH-SY5Y cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugate at 1/1000 dilution

Predicted band size: 92 kDa