Purified ab32513 at 1/40 dilution (2µg) immunoprecipitating Rb in Jurkat whole cell lysate.
Lane 1 (input): Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate 10µg
Lane 2 (+): ab32513 + Jurkat whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32513 in Jurkat whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 120 kDa

All lanes : Anti-Rb antibody [E182] (ab32513) at 1/1000 dilution (Purified)

Lane 1 : Jurkat (Human T cell leukemia T lymphocyte) prepared in RIPA lysis method whole cell lysate
Lane 2 : Jurkat (Human T cell leukemia T lymphocyte) prepared in 1% SDS Hot lysis method whole cell lysate

Lysates/proteins at 15 µg per lane.

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

Predicted band size: 106 kDa
Observed band size: 106-120 kDa why is the actual band size different from the predicted?

1% SDS Hot lysis method is preferred for this antibody.

Blocking Buffer and concentration: 5% NFDM/TBST

Immunocytochemistry analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labeling Rb with Purified ab32513 at 1:50 dilution (10 ?g/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.

Flow Cytometry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling Rb with Purified ab32513 at 1:70 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).

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: Rb knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: HEK293 whole cell lysate (20 µg)

 

Lanes 1 - 4: Merged signal (red and green). Green - ab32513 observed at 120 kDa. Red - loading control, ab8245, observed at 37 kDa.

 

ab32513 was shown to recognize Rb when Rb knockout samples were used, along with additional cross-reactive bands. Wild-type and Rb knockout samples were subjected to SDS-PAGE. Ab32513 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution 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 1/10000 dilution for 1 hour at room temperature before imaging.