Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: BIN1 (KO) knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: U-87MG whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab182562 observed at 50-65 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab182562 (unpurified) was shown to specifically react with BIN1 when BIN1 knockout samples were used. Wild-type and BIN1 knockout samples were subjected to SDS-PAGE. Ab182562 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1 µg/ml and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human ovarian carcinoma tissue sections labeling BIN1 with purified ab182562 at 1/500 dilution (0.52 µg/ml). Heat mediated antigen retrieval was performed using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

All lanes : Anti-BIN1 antibody [EPR13463] (ab182562) at 1/10000 dilution (Purified)

Lane 1 : Human brain lysates
Lane 2 : Human muscle lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 65 kDa
Observed band size: 50-65 kDa why is the actual band size different from the predicted?

The multiple bands are isoforms produced by alternative splicing

ab182562 (purified) at 1/20 dilution (1 µg) immunoprecipitating BIN1 in HeLa whole cell lysate.
Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10 µg
Lane 2 (+): ab182562 & HeLa whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab182562 in HeLa whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was at 1/1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling BIN1 with purified ab182562 at 1/30 dilution (10 µg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling BIN1 with purified ab182562 at 1:250 dilution (1 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunofluorescent analysis of HeLa cells labeling BIN1 with ab182562 (unpurified) at 1/250 and Goat anti rabbit IgG(Alexa Fluor®555) at 1/200.  Cells were fixed with -20℃ Acetone. Image at the right stained with DAPI.

Immunoprecipitation. ab182562 (unpurified) at 1/10000 staining BIN1 in U87-MG cell lysate immunoprecipitated using ab182562 at 1/50.
Lane 2: negative control.

Flow Cytometrical analysis of 2% paraformaldehyde fixed HeLa cells labeling BIN1 with ab182562 (unpurified) at 1/40.

Immunofluorescent analysis of U87-MG cells labeling BIN1 with ab182562 (unpurified) at 1/250 and Goat anti rabbit IgG(Alexa Fluor®555) at 1/200. Cells were fixed with 4% paraformaldehyde. Image at the right stained with DAPI.

Immunohistochemical analysis of paraffin embedded Human brain tissue labeling BIN1 with ab182562 (unpurified) at 1/100.

Immunohistochemical analysis of paraffin embedded Human clear cell carcinoma of kidney tissue labeling BIN1 with ab182562 (unpurified) at 1/100.

Immunohistochemical analysis of paraffin embedded Human skeletal muscle tissue labeling BIN1 with ab182562 (unpurified) at 1/100.

All lanes : Anti-BIN1 antibody [EPR13463] (ab182562) at 1/5000 dilution (unpurified)

Lane 1 : Human skeletal muscle
Lane 2 : Human fetal kidney

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG HRP (ab136636) at 1/500 dilution

Predicted band size: 65 kDa
Additional bands at: 56, 65, 70 kDa. We are unsure as to the identity of these extra bands.

All lanes : Anti-BIN1 antibody [EPR13463] (ab182562) at 1/5000 dilution (unpurified)

Lane 1 : A431 cell lysate
Lane 2 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 65 kDa
Additional bands at: 65, 70 kDa. We are unsure as to the identity of these extra bands.