Anti-Rb antibody [EPR17512] KO tested (ab181616)

Anti-Rb antibody EPR17512 is a rabbit monoclonal antibody that is used in Rb Western blot, IHC, ICC/IF, Flow cytometry, IP. Suitable for Human, Mouse and other species.

- Specificity confirmed with Rb1 knockout cell line validation
- Specificity and sensitivity confirmed in IHC with multi-tissue microarray (TMA) validation
- Cited in over 80 publications
- Sample sizes available

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Images

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rb antibody [EPR17512] (AB181616), expandable thumbnail

Publications

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal

Immunogen

The exact immunogen used to generate this antibody is proprietary information.

Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	IP	WB	ICC/IF	Flow Cyt (Intra)	IHC-P
Human	Tested	Tested	Tested	Tested	Tested
Mouse	Expected	Tested	Expected	Expected	Tested
African green monkey	Expected	Tested	Expected	Expected	Expected

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/80	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Expected
Expected

Species	Dilution info	Notes
Species Mouse, African green monkey	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/2000	Notes For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here. Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species African green monkey	Dilution info 1/2000	Notes For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here.
Species Human	Dilution info 1/2000	Notes For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here.

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/500	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse, African green monkey	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info -	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse, African green monkey	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/1000	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Human	Dilution info 1/1000	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Expected
Expected

Species	Dilution info	Notes
Species African green monkey	Dilution info Use at an assay dependent concentration.	Notes -

Associated Products

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Target data

See full target information RB1

Function

Tumor suppressor that is a key regulator of the G1/S transition of the cell cycle (PubMed:10499802). The hypophosphorylated form binds transcription regulators of the E2F family, preventing transcription of E2F-responsive genes (PubMed:10499802). Both physically blocks E2Fs transactivating domain and recruits chromatin-modifying enzymes that actively repress transcription (PubMed:10499802). Cyclin and CDK-dependent phosphorylation of RB1 induces its dissociation from E2Fs, thereby activating transcription of E2F responsive genes and triggering entry into S phase (PubMed:10499802). RB1 also promotes the G0-G1 transition upon phosphorylation and activation by CDK3/cyclin-C (PubMed:15084261). Directly involved in heterochromatin formation by maintaining overall chromatin structure and, in particular, that of constitutive heterochromatin by stabilizing histone methylation. Recruits and targets histone methyltransferases SUV39H1, KMT5B and KMT5C, leading to epigenetic transcriptional repression. Controls histone H4 'Lys-20' trimethylation. Inhibits the intrinsic kinase activity of TAF1. Mediates transcriptional repression by SMARCA4/BRG1 by recruiting a histone deacetylase (HDAC) complex to the c-FOS promoter. In resting neurons, transcription of the c-FOS promoter is inhibited by BRG1-dependent recruitment of a phospho-RB1-HDAC1 repressor complex. Upon calcium influx, RB1 is dephosphorylated by calcineurin, which leads to release of the repressor complex (By similarity). (Microbial infection) In case of viral infections, interactions with SV40 large T antigen, HPV E7 protein or adenovirus E1A protein induce the disassembly of RB1-E2F1 complex thereby disrupting RB1's activity.

Alternative names

Retinoblastoma-associated protein, p105-Rb, p110-RB1, pRb, pp110, Rb, RB1

Recommended products

Anti-Rb antibody EPR17512 is a rabbit monoclonal antibody that is used in Rb Western blot, IHC, ICC/IF, Flow cytometry, IP. Suitable for Human, Mouse and other species.

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal
Immunogen: The exact immunogen used to generate this antibody is proprietary information.
Clone number: EPR17512
Purification technique: Affinity purification Protein A
Concentration

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage duration: 1-2 weeks
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: -20°C
Aliquoting information: Upon delivery aliquot
Storage information: Avoid freeze / thaw cycle

Notes

Product Specifications
Anti-Rb antibody [EPR17512] (ab181616) was developed by Abcam using patented rabbit monoclonal antibody technology and is validated for use in Flow Cyt (Intra), ICC/IF, IHC-P, IP, WB in african green monkey, human, mouse samples.
Anti-Rb antibody [EPR17512] (ab181616) specifically detects Rb (UniProt ID: P13405; Molecular weight: 105kDa) and is sold in a convenient trial size to enable initial testing (20 µL) and larger sizes for subsequent scaling up experiments (100 µL and 1 mL).

Quality and Validation
Abcam's high quality manufacturing and validation processes ensure Anti-Rb antibody [EPR17512] (ab181616) has high sensitivity and specificity alongside high lot-to-lot consistency and reproducibility.
The specificity of Anti-Rb antibody [EPR17512] (ab181616) has been confirmed by testing in knockout samples.
Anti-Rb antibody [EPR17512] (ab181616) has been cited over 83 times in peer reviewed journals and is trusted by the scientific community.

Related Products
Antibody clone EPR17512 is also available pre-conjugated to a variety of labels for your convenience - Alexa Fluor® 647, Alexa Fluor® 594, Alexa Fluor® 488, Alexa Fluor® 555 (ab300158, ab300670, ab300671, ab300672).

Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

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 batch production

For more information, read more on recombinant antibodies.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
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20 product images

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) (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) (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, Anti-GAPDH antibody [6C5] - 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 Anti-GAPDH antibody [6C5] - Loading Control 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 Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-Rb antibody [EPR17512] (ab181616)
Predicted band size: 106 kDa
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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab181616 in MCF7 whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
All lanes: Immunoprecipitation - Anti-Rb antibody [EPR17512] (ab181616)
Predicted band size: 106 kDa
Exposure time: 8s
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) (Goat Anti-Rabbit IgG H&L (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 Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed 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 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.
Flow Cytometry (Intracellular) - Anti-Rb antibody [EPR17512] (ab181616)
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)
Western blot - Anti-Rb antibody [EPR17512] (ab181616)
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.
All lanes: Western blot - 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 at 20 µg with 5% NFDM/TBST
Lane 2: HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates prepared in 1%SDS Hot lysis method at 20 µg with 5% NFDM/TBST
Lane 3: K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates prepared in RIPA lysis method at 20 µg with 5% NFDM/TBST
Lane 4: K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates prepared in 1%SDS Hot lysis method at 20 µg with 5% NFDM/TBST
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/1000 dilution
Predicted band size: 106 kDa
Western blot - Anti-Rb antibody [EPR17512] (ab181616)
Blocking/Dilution buffer: 5% NFDM/TBST.
The lysates were all prepared using 1%SDS Hot lysis method.
All lanes: Western blot - Anti-Rb antibody [EPR17512] (ab181616) at 1/20000 dilution
Lane 1: Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate at 20 µg
Lane 2: K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate at 20 µg
Lane 3: WEHI-3 (Mouse leukemia) whole cell lysate at 20 µg
Lane 4: COS-1 (African green monkey kidney fibroblast-like cell line) whole cell lysate at 20 µg
Lane 5: MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg
Secondary
All lanes: Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution
Predicted band size: 106 kDa, 42 kDa
Observed band size: 105 kDa, 42 kDa
Exposure time: 5s
Western blot - Anti-Rb antibody [EPR17512] (ab181616)
Blocking/Dilution buffer: 5% NFDM/TBST.
The lysates were all prepared using 1%SDS Hot lysis method.
All lanes: Western blot - Anti-Rb antibody [EPR17512] (ab181616) at 1/10000 dilution
Lane 1: F9 (Mouse embyro testicular cancer cell line) whole cell lysate at 10 µg
Lane 2: Mouse brain lysate at 10 µg
Lane 3: Mouse lung lysate at 10 µg
Secondary
All lanes: Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution
Predicted band size: 106 kDa
Observed band size: 105 kDa
Exposure time: 1min
Western blot - Anti-Rb antibody [EPR17512] (ab181616)
Blocking/Dilution buffer: 5% NFDM/TBST.
The lysates were all prepared using 1%SDS Hot lysis method.
All lanes: Western blot - Anti-Rb antibody [EPR17512] (ab181616) at 1/2000 dilution
All lanes: Human fetal brain lysate at 10 µg
Secondary
All lanes: Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution
Predicted band size: 106 kDa, 188 kDa, 82 kDa
Observed band size: 105 kDa, 188 kDa, 82 kDa
Exposure time: 1min
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) (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) (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) (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) (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) (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) (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)
Western blot: Anti-RB1 antibody [EPR17512] (ab181616) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control 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] (ab181616) 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
Performed under reducing conditions.
Observed band size: 106 kDa
Western blot - Anti-Rb antibody [EPR17512] (ab181616)
Blocking and diluting buffer and concentration: 5% NFDM/TBST.
In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (Anti-6X His tag® antibody [EPR20547] - ChIP Grade ab213204) staining at 1/5000 dilution.
In Western blot, Anti-Vinculin antibody [EPR8185] (Anti-Vinculin antibody [EPR8185] ab129002) staining at 1/10000 dilution.
In Western blot, Anti-Rb antibody [EPR17512-291] (ab181616) staining at 1/1000 dilution.
All lanes: Western blot - Anti-Rb (phospho S795) antibody [EPR28156-6] (Anti-Rb (phospho S795) antibody [EPR28156-6] ab321974) at 1/1000 dilution
Lane 1: 293T cells transfected with a human Rb expression vector containing a His-tag, whole cell lysate at 20 µg
Lane 2: 293T cells transfected with a human Rb (S795A mutation) expression vector containing a His-tag, whole cell lysate at 20 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Observed band size: 106 kDa, 124 kDa
Exposure time: 1s
Western blot - Anti-Rb antibody [EPR17512] (ab181616)
Blocking and diluting buffer and concentration: 5% NFDM/TBST.
To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.
This blot was developed using a high sensitivity ECL substrate.
The identity of the bands at approximately 40 kDa, 60 kDa and 280 kDa (in lanes 1-2) are unknown.
In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.
In Western blot, Anti-Rb antibody [EPR17512-291] (ab181616) staining at 1/1000 dilution.
All lanes: Western blot - Anti-Rb (phospho S795) antibody [EPR28156-6] (Anti-Rb (phospho S795) antibody [EPR28156-6] ab321974) at 1/1000 dilution
Lane 1: MCF7 (human breast adenocarcinoma epithelial cell) serum starvation for 48h, whole cell lysate (untreated membrane) at 50 µg
Lane 2: MCF7 serum starvation for 48h, then treated with 10% FBS for 20h, whole cell lysate (untreated membrane) at 50 µg
Lane 3: MCF7 serum starvation for 48h, then treated with 10% FBS for 20h, whole cell lysate (alkaline phosphatase treated membrane) at 50 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Developed using the ECL technique.
Observed band size: 106 kDa, 36 kDa
Exposure time: 92s
Western blot - Anti-Rb antibody [EPR17512] (ab181616)
Blocking and diluting buffer and concentration: 5% NFDM/TBST.
To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.
In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.
In Western blot, Anti-Rb antibody [EPR17512-291] (ab181616) staining at 1/1000 dilution.
All lanes: Western blot - Anti-Rb (phospho S795) antibody [EPR28156-6] (Anti-Rb (phospho S795) antibody [EPR28156-6] ab321974) at 1/1000 dilution
Lane 1: Jurkat (human T cell leukemia T lymphocyte from peripheral blood) serum starvation for 48h, whole cell lysate (untreated membrane) at 50 µg
Lane 2: Jurkat serum starvation for 48h, then treated with 10% FBS for 20h, whole cell lysate (untreated membrane) at 50 µg
Lane 3: Jurkat serum starvation for 48h, then treated with 10% FBS for 20h, whole cell lysate (alkaline phosphatase treated membrane) at 50 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Observed band size: 106 kDa, 36 kDa
Exposure time: 15s
Western blot - Anti-Rb antibody [EPR17512] (ab181616)
Blocking and diluting buffer and concentration: 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.

In Western blot, Anti-Rb antibody [EPR17512] (ab181616) staining at 1/1000 dilution.

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (Anti-6X His tag® antibody [EPR20547] - ChIP Grade ab213204) staining at 1/5000 dilution.
All lanes: Western blot - Anti-Rb (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] (Anti-Rb (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] ab320747) at 1/1000 dilution
Lane 1: 293T cells transfected with an empty vector containing a myc-his tag, whole cell lysate at 20 µg
Lane 2: 293T cells transfected with a human Rb1 expression vector containing a myc-his-tag, whole cell lysate at 2 µg
Lane 3: 293T cells transfected with a human Rb1(T252A, T56A, S608A, S780A, S807A) expression vector containing a myc-his-tag, whole cell lysate at 40 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Observed band size: 110 kDa, 36 kDa
Exposure time: 15s
Western blot - Anti-Rb antibody [EPR17512] (ab181616)
Blocking and diluting buffer and concentration: 5% NFDM/TBST.

Palbociclib is a CDK4/6 inhibitor. Treatment leads to a reduction in total Rb1 levels through G1 phase arrest.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.

In Western blot, Anti-Rb antibody [EPR17512] (ab181616) staining at 1/1000 dilution.
All lanes: Western blot - Anti-Rb (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] (Anti-Rb (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] ab320747) at 1/1000 dilution
Lane 1: Untreated Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate (untreated membrane) at 20 µg
Lane 2: Jurkat treated with 1uM palbociclib for 48 hours whole cell lysate (untreated membrane) at 20 µg
Lane 3: Untreated Jurkat whole cell lysate (alkaline phosphatase treated membrane) at 20 µg
Lane 4: Jurkat treated with 1uM palbociclib for 8 hours whole cell lysate (alkaline phosphatase treated membrane) at 20 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Observed band size: 110 kDa, 36 kDa
Exposure time: 37s
Western blot - Anti-Rb antibody [EPR17512] (ab181616)
Blocking and diluting buffer and concentration: 5% NFDM/TBST.

In Western blot, Anti-Rb (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] ab320747 was shown to bind specifically to RB1. Target of interest was observed at 100 kDa in wild-type HAP1 cell lysates (lane 1) with no signal observed at this size in RB1 knockout cell line (lane 2).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.

In Western blot, Anti-Rb antibody [EPR17512] (ab181616) staining at 1/1000 dilution.
All lanes: Western blot - Anti-Rb (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] (Anti-Rb (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] ab320747) at 1/1000 dilution
Lane 1: Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate (untreated membrane) at 20 µg
Lane 2: RB1 knockout HAP1 whole cell lysate (untreated membrane) at 20 µg
Lane 3: Wild-type HAP1 whole cell lysate (alkaline phosphatase treated membrane) at 20 µg
Lane 4: RB1 knockout HAP1 whole cell lysate (alkaline phosphatase treated membrane) at 20 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Performed under reducing conditions.
Observed band size: 110 kDa, 36 kDa
Exposure time: 37s
Western blot - Anti-Rb antibody [EPR17512] (ab181616)
Western blot: Rabbit monoclonal [EPR17512] to Rb ab181616 staining at 1/1000 dilution, shown in green; Mouse anti alpha Tubulin (Anti-alpha Tubulin antibody [DM1A] - Loading Control 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] (ab181616) at 1/1000 dilution
Lane 1: Wild-type HCT 116 at 20 µg
Lane 2: Western blot - Human RB1 knockout HCT116 cell line (Human RB1 knockout HCT116 cell line ab286657) at 20 µg
Lane 3: Wild-type A549 ab288558 at 20 µg
Lane 4: Western blot - Human RB1 knockout A549 cell line (Human RB1 knockout A549 cell line ab286470) 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
Performed under reducing conditions.
Predicted band size: 106 kDa
Observed band size: 106 kDa