All lanes : Anti-Junctional Adhesion Molecule 1/JAM-A antibody [EPR23246-275] (ab270446) at 1/1000 dilution

Lane 1 : bEnd.3 (mouse brain endothelioma), whole cell lysate
Lane 2 : Mouse testis tissue lysate
Lane 3 : Mouse brain tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 dilution

Predicted band size: 32 kDa
Observed band size: 32,35 kDa why is the actual band size different from the predicted?

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

Bands of 35Da and 32KDa respectively represent the glycosylated form and non-glycosylated form.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 10753840, 10698320 ).

Negative control: brain (PMID: 10698320).

Exposure time: 10 seconds

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized bEnd.3 cells labelling Junctional Adhesion Molecule 1/JAM-A with ab270446 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing membranous staining in bEnd.3 cell line ab195889 Anti-alpha Tubulin antibody (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1000 dilution.

Flow cytometric analysis of Neuro-2a (Mouse neuroblastoma neuroblast, Left) / bEnd.3 (Mouse brain endothelioma, Right) cells labelling Junctional Adhesion Molecule 1/JAM-A with ab270446 at 1/600 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. Gated on viable cells.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab270446).

Junctional Adhesion Molecule 1/JAM-A was immunoprecipitated from 0.35 mg bEnd.3 (mouse brain endothelioma), whole cell lysate with ab270446 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab270446 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: bEnd.3 (mouse brain endothelioma), whole cell lysate 10 ug

Lane 2: ab270446 IP in bEnd.3 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab270446 in bEnd.3 whole cell lysate

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

Exposure time: 10 seconds

 

Anti-Junctional Adhesion Molecule 1/JAM-A antibody [EPR23246-275] (ab270446) at 1/1000 dilution + Recombinant Mouse JAM-A Fc Chimera Protein (aa27-242)

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 32 kDa
Observed band size: 53 kDa why is the actual band size different from the predicted?

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

Exposure time: 10 seconds

Anti-Junctional Adhesion Molecule 1/JAM-A antibody [EPR23246-275] (ab270446) at 1/1000 dilution + RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 dilution

Predicted band size: 32 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?

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

Bands of 35Da and 32KDa respectively represent the glycosylated form and non-glycosylated form.

The expression profile: molecular weight observed is consistent with what has been described in the literature (PMID: 10753840, 10698320 ).

Exposure time: 3 minutes