Recombinant Anti-Rb antibody [EPR17512] (ab181616)

Immunohistochemical analysis of paraffin-embedded Human lung tissue labeling Rb with ab181616 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on Human lung is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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 - ab181616 observed at 120 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab181616 was shown to recognize Rb in wild-type HAP1 cells as signal was lost at the expected MW in Rb knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and RB1 knockout samples were subjected to SDS-PAGE. Ab181616 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/2000 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.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MCF7 (Human breast adenocarcinoma cell line) cells labeling Rb with ab181616 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing nuclear staining on MCF7 cells. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
-ve control 1: ab181616 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

ab181616 staining Rbin the human cell line MCF-7 (human breast carcinoma) by intracellular flow cytometry. Cells were fixed with 4% paraformaldehyde, permeabilized with 90% methanol and the sample was incubated with the primary antibody at a dilution of 1/140. A goat anti rabbit IgG (Alexa Fluor^® 488) at a dilution of 1/2000 was used as the secondary antibody. 

Isoytype control: Rabbit monoclonal IgG (Black) 

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue) 

Exposure time:

Lane 1 and 2: 100 seconds
Lane 3 and 4: 10 seconds

We recommend to use 1%SDS Hot lysis method to get clear band.
We are unsure how to define the extra bands.

Rb was immunoprecipitated from 1mg of MCF7 (Human breast adenocarcinoma cell line) whole cell lysate with ab181616 at 1/80 dilution. Western blot was performed from the immunoprecipitate using ab181616 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1: MCF7 whole cell lysate 10 µg (Input). Lane 2: ab181616 IP in MCF7 whole cell lysate. Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab181616 in MCF7 whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Blocking/Dilution buffer: 5% NFDM/TBST.

The lysates were all prepared using 1%SDS Hot lysis method.

Blocking/Dilution buffer: 5% NFDM/TBST.

The lysates were all prepared using 1%SDS Hot lysis method.

Blocking/Dilution buffer: 5% NFDM/TBST.

The lysates were all prepared using 1%SDS Hot lysis method.

Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labeling Rb with ab181616 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on cancer cells of Human breast cancer is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Mouse lung tissue labeling Rb with ab181616 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on mouse lung is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Mouse cerebral cortex tissue labeling Rb with ab181616 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on neuron of mouse cerebral cortex is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.