Anti-Neogenin antibody [EPR25220-56]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Neogenin antibody [EPR25220-56] (AB302542)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized A549 (human lung carcinoma epithelial cell) cells labelling Neogenin with ab302542 at 1/50 (10.08 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/mL dilution (Green). Confocal image showing membranal and cytoplasmic staining in A549 cell line is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Neogenin antibody [EPR25220-56] (AB302542)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized SW480 (human colorectal adenocarcinoma epithelial cell) cells labelling Neogenin with ab302542 at 1/50 (10.08 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/mL dilution (Green). Confocal image showing membranal and cytoplasmic staining in SW480 cell line is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• WB

Lab

Western blot - Anti-Neogenin antibody [EPR25220-56] (AB302542)

Western blot : Anti-NEO1 antibody [EPR25220-56] (ab302542) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab302542 was shown to bind specifically to NEO1. A band was observed at 110-200 kDa in wild-type A549 cell lysates with no signal observed at this size in NEO1 knockout cell line ab282825 (knockout cell lysate ab283049). To generate this image, wild-type and NEO1 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1% Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

All lanes:

Western blot - Anti-Neogenin antibody [EPR25220-56] (ab302542)

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

NEO1 knockout A549 cell lysate at 20 µg

Lane 3:

SH-SY5Y cell lysate at 20 µg

Lane 4:

Western blot at 20 µg

Observed band size: 110-200 kDa

true

• WB

Supplier Data

Western blot - Anti-Neogenin antibody [EPR25220-56] (AB302542)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-Neogenin antibody [EPR25220-56] (ab302542) at 1/1000 dilution

Lane 1:

His-tagged human Neogenin recombinant protein 10 ng

Lane 2:

His-tagged human DCC recombinant protein 10 ng

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 1/100000 dilution

Observed band size: 60 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-Neogenin antibody [EPR25220-56] (AB302542)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The expression molecular weight observed is consistent with what has been described in the literature (PMID : 30858446, 33088218).

All lanes:

Western blot - Anti-Neogenin antibody [EPR25220-56] (ab302542) at 1/1000 dilution

Lane 1:

SW480 (human colorectal adenocarcinoma epithelial cell) whole cell lysate 20 µg

Lane 2:

Caco-2 (human colorectal adenocarcinoma epithelial cell) whole cell lysate 20 µg

Lane 3:

Untreated A549 (human lu carcinoma epithelial cell) whole cell lysate 20 µg

Lane 4:

A549 whole cell lysate treated with Protein Deglycosylation MIX II, 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

Observed band size: 210 kDa,160 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-Neogenin antibody [EPR25220-56] (AB302542)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-Neogenin antibody [EPR25220-56] (ab302542) at 1/1000 dilution

Lane 1:

A549 (human lu carcinoma epithelial cell) transfected with scrambled siRNA control, whole cell lysate 20 µg

Lane 2:

A549 transfected with siRNA specifically targeting Neogenin), whole cell lysate 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

Observed band size: 210 kDa

false

Exposure time: 26s