Anti-Neogenin 抗体 [EPR25220-56] (ab302542)

All lanes : Anti-Neogenin antibody [EPR25220-56] (ab302542) at 1/1000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : NEO1 knockout A549 cell lysate
Lane 3 : SH-SY5Y cell lysate
Lane 4 : Neuro-2a cell lysate

Lysates/proteins at 20 µg per lane.

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 160 kDa
Observed band size: 110-200 kDa why is the actual band size different from the predicted?

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

Lane 1 : A549 (human lung carcinoma epithelial cell) transfected with scrambled siRNA control, whole cell lysate
Lane 2 : A549 transfected with siRNA specifically targeting Neogenin), whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 160 kDa
Observed band size: 210 kDa why is the actual band size different from the predicted?


Exposure time: 26 seconds

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

All lanes : Anti-Neogenin antibody [EPR25220-56] (ab302542) at 1/1000 dilution

Lane 1 : SW480 (human colorectal adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : Caco-2 (human colorectal adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : Untreated A549 (human lung carcinoma epithelial cell) whole cell lysate
Lane 4 : A549 whole cell lysate treated with Protein Deglycosylation MIX II

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 160 kDa
Observed band size: 210, 160 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking and dilution 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 : 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 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 160 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking and dilution buffer and concentration: 5% NFDM/TBST. 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized SW480 (human colorectal adenocarcinoma epithelial cell) cells labeling Neogenin with AB302542 at 1/50 (10.08 µg/mL) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^®488) preabsorbed antibody at 1/1000 dilution (2 µg/mL) (Green). Confocal image showing membranal and cytoplasmic staining in SW480 cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (2.5 µg/mL) (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody control: Primary diluent was used instead of primary antibody, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preabsorbed at 1/1000 dilution (2 µg/mL).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized A549 (human lung carcinoma epithelial cell) cells labeling Neogenin with AB302542 at 1/50 (10.08 µg/mL) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preabsorbed antibody at 1/1000 dilution (2 µg/mL) (Green). Confocal image showing membranal and cytoplasmic staining in A549 cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (2.5 µg/mL) (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody control: Primary diluent was used instead of primary antibody, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^_® 488) preabsorbed at 1/1000 dilution (2 µg/mL).

Neogenin was immunoprecipitated from 0.35 mg A549 (human lung carcinoma epithelial cell) whole cell lysate with AB302542 at 1/30 dilution (2 µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using AB302542 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: A549 (human lung carcinoma epithelial cell) whole cell lysate 10 µg (Inset)

Lane 2: AB302542 IP in A549 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of AB302542 in A549 whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 3 minutes

 Observed MW (kDa): 210