Anti-Sortilin/NT3 antibody [EPR15010]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Sortilin/NT3 antibody [EPR15010] (AB188586)

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.

• WB

Lab

Western blot - Anti-Sortilin/NT3 antibody [EPR15010] (AB188586)

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:

Western blot - Anti-Sortilin/NT3 antibody [EPR15010] (ab188586)

Predicted band size: 92 kDa

false

• WB

Lab

Western blot - Anti-Sortilin/NT3 antibody [EPR15010] (AB188586)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Sortilin/NT3 antibody [EPR15010] (ab188586) at 1/1000 dilution

Lane 1:

SH-SY5Y whole cell lysate at 15 µg

Lane 2:

Human hippocampus tissue lysate at 15 µg

Lane 3:

Human fetal brain tissue lysate at 15 µg

Lane 4:

Mouse brain tissue lysate at 15 µg

Lane 5:

3T3-L1 whole cell lysate at 15 µg

Lane 6:

Rat brain tissue lysate at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 92 kDa

Observed band size: 92 kDa

false

Exposure time: 30s

• WB

Lab

Western blot - Anti-Sortilin/NT3 antibody [EPR15010] (AB188586)

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.

All lanes:

Western blot - Anti-Sortilin/NT3 antibody [EPR15010] (ab188586) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human SORT1 (Sortilin/NT3) knockout HeLa cell lysate (<a href='/en-us/products/cell-lysates/human-sort1-sortilin-nt3-knockout-hela-cell-lysate-ab257696'>ab257696</a>) at 20 µg

Secondary

Lanes 1 - 2:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Lanes 1 - 2:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Predicted band size: 92 kDa

Observed band size: 100 kDa

false

• WB

Supplier Data

Western blot - Anti-Sortilin/NT3 antibody [EPR15010] (AB188586)

All lanes:

Western blot - Anti-Sortilin/NT3 antibody [EPR15010] (ab188586) at 1/2000 dilution

Lane 1:

SW480 cell lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Lane 3:

SH-SY5Y cell lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 92 kDa

false