Antibodies, Proteins, Kits and Reagents for Life Science

360 577 ₸ Product size

100 µl 360 577 ₸

Order now and get it on Tuesday March 02, 2021

Key features and details

Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
Rabbit monoclonal [EPR17512] to Rb
Suitable for: Flow Cyt, WB, IP, ICC/IF, IHC-P
Knockout validated
Reacts with: Mouse, Human, African green monkey

Product name

Anti-Rb antibody [EPR17512]
See all Rb primary antibodies
Description

Rabbit monoclonal [EPR17512] to Rb
Host species

Rabbit

Tested Applications & Species

Application	Species
Flow Cyt	Human
ICC/IF	Human
IHC-P	Mouse Human
IP	Human
WB	Mouse Human

See all applications and species data

Immunogen

Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
Positive control
- WB: Jurkat, Hek294, K562, WEHI-3, COS-1, MCF7 and F9 whole cell lysates; Mouse brain and lung lysates; Human fetal brain lysate. IHC-P: Human lung, Human breast cancer, Mouse lung and Mouse cerebral cortex tissues. ICC/IF: MCF7 cells. IP: MCF7 whole cell lysate.
General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
For more information see here.
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.
We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.
Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.

Form

Liquid
Storage instructions

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
Storage buffer

Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol, 0.05% BSA
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

EPR17512
Isotype

IgG
Research areas

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - 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.
Western blot - Anti-Rb antibody [EPR17512] (ab181616)

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.
Immunocytochemistry/ Immunofluorescence - Anti-Rb antibody [EPR17512] (ab181616)

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.
Flow Cytometry - Anti-Rb antibody [EPR17512] (ab181616)

ab181616 staining Rb in the human cell line MCF-7(human breast carcinoma) by 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)
Western blot - Anti-Rb antibody [EPR17512] (ab181616)

All lanes : Anti-Rb antibody [EPR17512] (ab181616) at 1/1000 dilution

Lane 1 : HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates prepared in RIPA lysis method with 5% NFDM/TBST
Lane 2 : HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates prepared in 1%SDS Hot lysis method with 5% NFDM/TBST
Lane 3 : K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates prepared in RIPA lysis method with 5% NFDM/TBST
Lane 4 : K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates prepared in 1%SDS Hot lysis method with 5% NFDM/TBST

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution

Predicted band size: 105 kDa

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.
Immunoprecipitation - Anti-Rb antibody [EPR17512] (ab181616)

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.
Western blot - Anti-Rb antibody [EPR17512] (ab181616)

All lanes : Anti-Rb antibody [EPR17512] (ab181616) at 1/20000 dilution

Lane 1 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate
Lane 2 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate
Lane 3 : WEHI-3 (Mouse leukemia) whole cell lysate
Lane 4 : COS-1 (African green monkey kidney fibroblast-like cell line) whole cell lysate
Lane 5 : MCF7 (Human breast adenocarcinoma cell line) whole 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: 105 kDa
Observed band size: 105 kDa

Exposure time: 5 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The lysates were all prepared using 1%SDS Hot lysis method.
Western blot - Anti-Rb antibody [EPR17512] (ab181616)

All lanes : Anti-Rb antibody [EPR17512] (ab181616) at 1/10000 dilution

Lane 1 : F9 (Mouse embyro testicular cancer cell line) whole cell lysate
Lane 2 : Mouse brain lysate
Lane 3 : Mouse lung lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 105 kDa
Observed band size: 105 kDa

Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

The lysates were all prepared using 1%SDS Hot lysis method.
Western blot - Anti-Rb antibody [EPR17512] (ab181616)

Anti-Rb antibody [EPR17512] (ab181616) at 1/2000 dilution + Human fetal brain lysate at 10 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 105 kDa
Observed band size: 105 kDa

Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

The lysates were all prepared using 1%SDS Hot lysis method.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rb antibody [EPR17512] (ab181616)

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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rb antibody [EPR17512] (ab181616)

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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rb antibody [EPR17512] (ab181616)

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.
Anti-Rb antibody [EPR17512] (ab181616)

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