Anti-Rb (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] KO Tested (ab320747)

Knockout Tested Rabbit Recombinant Multiclonal RB phospho T252 + T356 + S608 + S780 + S807 antibody. Suitable for Dot, IHC-P, WB, IP and reacts with Synthetic peptide - Human, Mouse, Rat, Human, Transfected cell lysate - Human samples.

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Images

Western blot - Anti-Rb (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] (AB320747), expandable thumbnail

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Multiclonal

Immunogen

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

Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	Dot	IHC-P	WB	IP	ICC/IF
Human	Expected	Tested	Tested	Tested	Not recommended
Mouse	Expected	Tested	Expected	Expected	Not recommended
Rat	Expected	Tested	Expected	Expected	Not recommended
Synthetic peptide - Human	Tested	Not recommended	Not recommended	Not recommended	Not recommended
Transfected cell lysate - Human	Not recommended	Not recommended	Tested	Not recommended	Not recommended

Tested
Tested

Species	Dilution info	Notes
Species Synthetic peptide - Human	Dilution info 1/1000	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse, Rat, Human	Dilution info Use at an assay dependent concentration.	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Transfected cell lysate - Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/500	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Rat	Dilution info 1/500	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Human	Dilution info 1/500	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Not recommended
Not recommended

Species	Dilution info	Notes
Species Synthetic peptide - Human, Transfected cell lysate - Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/1000	Notes -
Species Transfected cell lysate - Human	Dilution info -	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse, Rat	Dilution info Use at an assay dependent concentration.	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Synthetic peptide - Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/30	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse, Rat	Dilution info Use at an assay dependent concentration.	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Synthetic peptide - Human, Transfected cell lysate - Human	Dilution info -	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Human, Mouse, Rat, Synthetic peptide - Human, Transfected cell lysate - Human	Dilution info -	Notes -

Target data

See full target information RB1 phospho T252 + T356 + S608 + S780 + S807

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

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Multiclonal
Immunogens: The exact immunogen used to generate this antibody is proprietary information.
Clone number: RM1144
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

This product is a recombinant multiclonal 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.

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

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

Retinoblastoma protein (Rb) also known as pRb is an important regulatory protein with a molecular weight of approximately 110 kDa. It is mainly expressed in the nucleus of cells. Rb functions as a tumor suppressor by controlling the cell cycle progression from G1 to S phase. Rb becomes active when it is dephosphorylated allowing it to bind and inhibit E2F transcription factors consequently preventing the transcription of genes essential for S phase entry.

Biological function summary

Retinoblastoma protein influences cellular proliferation differentiation and apoptosis. It acts within a larger protein complex modulating various cellular responses. When functional Rb halts uncontrolled cell division important for maintaining normal tissue homeostasis. In its phosphorylated state known as phospho-Rb or phospho-Rb E182 it loses its regulatory capabilities which can lead to unrestrained cell cycle progression.

Pathways

Several involve the retinoblastoma protein. One key pathway is the p53 pathway which Rb interacts with to influence cellular outcomes like cell-cycle arrest and apoptosis. Rb cooperates with proteins like p21 to implement these processes. Additionally it is involved in the cyclin D-dependent kinase 4 (CDK4) pathway which modulates its phosphorylation state influencing binding interactions and cell cycle control.

Associated diseases and disorders

Dysregulation of retinoblastoma protein is commonly associated with cancer particularly retinoblastoma and breast cancer. In retinoblastoma mutations in the Rb gene directly lead to uncontrolled cell proliferation due to the absence of functional Rb protein. Additionally Rb's connection to the E2F family of transcription factors can become disrupted contributing to oncogenesis in other cancers.

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:
Terms & Conditions.

10 product images

Western blot - Anti-Rb (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] (ab320747)
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] (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] (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
Dot Blot - Anti-Rb (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] (ab320747)
Dot blot analysis of Rb (phospho T252 + T356 + S608 + S780 + S807) using ab320747 at 1:1000 (0.511 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1:100,000 dilution.

Lane 1: Human Rb1 non-phospho peptide
Lane 2: Human Rb1 (phospho T252) peptide
Lane 3: Human Rb1 non-phospho peptide
Lane 4: Human Rb1 (phospho T356) peptide
Lane 5: Human Rb1 non-phospho peptide
Lane 6: Human Rb1 (phospho S608) peptide
Lane 7: Human Rb1 non-phospho peptide
Lane 8: Human Rb1 (phospho S780) peptide
Lane 9: Human Rb1 non-phospho peptide
Lane 10: Human Rb1 (phospho S807) peptide

Exposure time: 180 seconds.

Blocking and diluting buffer and concentration: 5% NFDM/TBST.
All lanes: Dot Blot - Anti-Rb (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] (ab320747) at 1/1000 dilution
Lanes 1, 3, 5, 7 and 9: Human Rb1 non-phospho peptide
Lane 2: Human Rb1 (phospho T252) peptide
Lane 4: Human Rb1 (phospho T356) peptide
Lane 6: Human Rb1 (phospho S608) peptide
Lane 8: Human Rb1 (phospho S780) peptide
Lane 10: Human Rb1 (phospho S807) peptide
Secondary
All lanes: Dot Blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Exposure time: 180s
Immunoprecipitation - Anti-Rb (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] (ab320747)
Rb (phospho T252 + T356 + S608 + S780 + S807) was immunoprecipitated from 0.35 mg Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate with ab320747 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab320747 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.

Lane 1: Jurkat whole cell lysate
Lane 2: ab320747 IP in Jurkat whole cell lysate
Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab320747 in Jurkat whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 3 seconds.
All lanes: Immunoprecipitation - Anti-Rb (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] (ab320747) at 1/30 dilution
All lanes: Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate
Secondary
All lanes: Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/5000 dilution
Exposure time: 3s
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rb (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] (ab320747)
Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labeling Rb (phospho T252 + T356 + S608 + S780 + S807) with ab320747 at 1/2000 (0.256 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) rat spleen without alkaline phosphatase treatment, no staining on (B) rat spleen with alkaline phosphatase treatment. The section was incubated with ab320747 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rb (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] (ab320747)
Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling Rb (phospho T252 + T356 + S608 + S780 + S807) with ab320747 at 1/2000 (0.256 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) mouse spleen without alkaline phosphatase treatment, no staining on (B) mouse spleen with alkaline phosphatase treatment. The section was incubated with ab320747 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rb (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] (ab320747)
Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling Rb (phospho T252 + T356 + S608 + S780 + S807) with ab320747 at 1/2000 (0.256 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) mouse colon without alkaline phosphatase treatment, no staining on (B) mouse colon with alkaline phosphatase treatment. The section was incubated with ab320747 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rb (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] (ab320747)
Immunohistochemical analysis of paraffin-embedded Human cervical carcinoma tissue labeling Rb (phospho T252 + T356 + S608 + S780 + S807) with ab320747 at 1/500 (1.022 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) human cervical carcinoma without alkaline phosphatase treatment, no staining on (B) human cervical carcinoma with alkaline phosphatase treatment. The section was incubated with ab320747 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rb (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] (ab320747)
Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Rb (phospho T252 + T356 + S608 + S780 + S807) with ab320747 at 1/500 (1.022 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) human colon without alkaline phosphatase treatment, no staining on (B) human colon with alkaline phosphatase treatment. The section was incubated with ab320747 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins
Western blot - Anti-Rb (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] (ab320747)
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] (Anti-Rb antibody [EPR17512] ab181616) staining at 1/1000 dilution.
All lanes: Western blot - 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 (phospho T252 + T356 + S608 + S780 + S807) antibody [RM1144] (ab320747)
Blocking and diluting buffer and concentration: 5% NFDM/TBST.

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