Anti-Rb antibody [EPR17512] - BSA and Azide free

• WB

Lab

Western blot - Anti-Rb antibody [EPR17512] - BSA and Azide free (AB218526)

This data was developed using the same antibody clone in a different buffer formulation (abAB181616).

Western blot : Anti-RB1 antibody [EPR17512] (ab181616) 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, ab181616 was shown to bind specifically to RB1. A band was observed at 106 kDa in wild-type A549 cell lysates with no signal observed at this size in RB1 knockout cell line. To generate this image, wild-type and RB1 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 5 % 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:

Western blot - Anti-Rb antibody [EPR17512] (<a href='/en-us/products/primary-antibodies/rb-antibody-epr17512-ab181616'>ab181616</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 40 µg

Lanes 2 - 3:

RB1 knockout A549 cell lysate at 40 µg

Lanes 4 - 5:

HeLa cell lysate at 20 µg

Lane 6:

Wild-type HAP1 cell lysate at 20 µg

Lane 7:

RB1 knockout HAP1 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 106 kDa

false

• WB

Lab

Western blot - Anti-Rb antibody [EPR17512] - BSA and Azide free (AB218526)

This data was developed using the same antibody clone in a different buffer formulation (ab181616).

Western blot : Rabbit monoclonal [EPR17512] to Rb ab181616 staining at 1/1000 dilution, shown in green; Mouse anti alpha Tubulin (ab7291) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 106 kDa in Wild-type HCT 116 cell lysates with no signal observed at this size in RB1 knockout HCT 116 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Rb antibody [EPR17512] (<a href='/en-us/products/primary-antibodies/rb-antibody-epr17512-ab181616'>ab181616</a>) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 at 20 µg

Lane 2:

Western blot - Human RB1 knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-rb1-knockout-hct116-cell-line-ab286657'>ab286657</a>) at 20 µg

Lane 3:

Wild-type A549 ab288558 at 20 µg

Lane 4:

Western blot - Human RB1 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-rb1-knockout-a549-cell-line-ab286470'>ab286470</a>) at 20 µg

Lane 5:

HEK-293 at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 106 kDa

Observed band size: 106 kDa

false

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Rb antibody [EPR17512] - BSA and Azide free (AB218526)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181616).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Rb antibody [EPR17512] - BSA and Azide free (AB218526)

ab181616 staining Rb in 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)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181616).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rb antibody [EPR17512] - BSA and Azide free (AB218526)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181616).

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rb antibody [EPR17512] - BSA and Azide free (AB218526)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181616).

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rb antibody [EPR17512] - BSA and Azide free (AB218526)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181616).

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rb antibody [EPR17512] - BSA and Azide free (AB218526)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181616).

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

• IP

Supplier Data

Immunoprecipitation - Anti-Rb antibody [EPR17512] - BSA and Azide free (AB218526)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181616).

All lanes:

Immunoprecipitation - Anti-Rb antibody [EPR17512] (<a href='/en-us/products/primary-antibodies/rb-antibody-epr17512-ab181616'>ab181616</a>)

Predicted band size: 106 kDa

false

Exposure time: 8s