Anti-Nicastrin antibody [EPR2575Y]

• WB

Lab

Western blot - Anti-Nicastrin antibody [EPR2575Y] (AB68145)

Lanes 1-4 : Merged signal (red and green). Green - ab68145 observed at 100-125 kDa. Red - loading control ab8245 observed at 36 kDa.

ab68145 Anti-Nicastrin antibody [EPR2575Y] was shown to specifically react with Nicastrin in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266602 (knockout cell lysate ab258061) was used. Wild-type and Nicastrin knockout samples were subjected to SDS-PAGE. ab68145 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 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-Nicastrin antibody [EPR2575Y] (ab68145) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

NCSTN knockout HEK293T cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

HL-60 cell lysate at 20 µg

Secondary

All lanes:

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 1/10000 dilution

Predicted band size: 78 kDa

Observed band size: 100-125 kDa

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• WB

Unknown

Western blot - Anti-Nicastrin antibody [EPR2575Y] (AB68145)

All lanes:

Western blot - Anti-Nicastrin antibody [EPR2575Y] (ab68145) at 1/1000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

HL-60 cell lysate at 10 µg

Lane 3:

Jurkat cell lysate at 10 µg

Secondary

All lanes:

HRP conjugated Goat anti-Rabbit at 1/2000 dilution

Predicted band size: 78 kDa

Observed band size: 100-120 kDa,130-160 kDa

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• WB

Lab

Western blot - Anti-Nicastrin antibody [EPR2575Y] (AB68145)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. Exposure time : Lane 1, 4 : 180 seconds; Lane 2, 3 : 15 seconds. The bands observed is consistency with literature (PMID : 15322084). The lysates were freshly made and used for Western Blotting immediately to minimize protein degradation.

All lanes:

Western blot - Anti-Nicastrin antibody [EPR2575Y] (ab68145) at 1/1000 dilution

Lane 1:

C6 (rat glial tumor glial cell) whole cell lysate at 20 µg

Lane 2:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

Lane 3:

U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 20 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: 78 kDa

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