Cell Signaling Technology

Product Pathways - Cell Cycle / Checkpoint

PhosphoPlus® Rb (Ser780, Ser807/811) Antibody Kit #9306

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Phospho-Rb (Ser780) Antibody #9307 100 µl W IP E-P H R Mk 110 Rabbit
Phospho-Rb (Ser807/811) Antibody #9308 100 µl W IP IHC-P H R Mk (M) 110 Rabbit
Rb (4H1) Mouse mAb #9309 100 µl W IP IHC-P IF-IC F ChIP H Mk B Pg 110 Mouse IgG2a
Rb Control Proteins #9303 100 µl 76
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat
Anti-biotin, HRP-linked Antibody #7075 100 µl Goat
Anti-mouse IgG, HRP-linked Antibody #7076 100 µl Horse
20X LumiGLO® Reagent and 20X Peroxide #7003 5 ml each
Biotinylated Protein Ladder Detection Pack #7727 100 µl

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry  ChIP=Chromatin IP  E-P=ELISA (Peptide)
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  B=Bovine  Pg=Pig
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

Phospho-Rb (Ser780) and Phospho-Rb (Ser807/811) antibodies detect endogenous levels of Rb only when phosphorylated at Ser780 and Ser807/811, respectively. These antibodies do not cross-react with Rb phosphorylated at other sites. Rb (4H1) Mouse mAb detects endogenous levels of total Rb protein. The antibody does not cross-react with the Rb homologues p107 or p130, or with other proteins.

Western Blotting

Western Blotting

Western blot analysis of Rb Control Protein #9303 using Rb (4H1) mAb #9309.

Western Blotting

Western Blotting

Western blot analysis of Rb phosphorylation in human fibroblasts synchronized by serum deprivation using Phospho-Rb (Ser780) Antibody #9307 (upper) and Phospho-Rb (Ser807/811) Antibody #9308 (lower). Cells were synchronized for 24 hours then released by addition of serum and harvested at the times indicated. Cell cycle progression was verified by cyclin analysis and FACS. (Provided by John Boylan, Dupont/Merck, Delaware.)

Description

The PhosphoPlus® Rb (Ser780, Ser807/811) Antibody Kit provides reagents and protocols to investigate cell cycle progression within cells. The kit contains a total Rb antibody, two distinct phospho-Rb antibodies, and Rb control proteins along with secondary antibodies and reagents to perform up to 10 western blots.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues around Ser780 and Ser807/811 of human Rb. Antibodies are purified by protein A and peptide affinity chromatography. Rb (4H1) monoclonal antibody is produced by immunizing animals with Rb-C Fusion Protein #6022, which contains residues 701-928 of human Rb .

Background

The retinoblastoma tumor suppressor protein, Rb, regulates cell proliferation by controlling progression through the restriction point within the G1-phase of the cell cycle (1). Rb has three functionally distinct binding domains and interacts with critical regulatory proteins including the E2F family of transcription factors, c-Abl tyrosine kinase, and proteins with a conserved LXCXE motif (2-4). Cell cycle-dependent phosphorylation by a CDK inhibits Rb target binding and allows cell cycle progression (5). Rb inactivation and subsequent cell cycle progression likely requires an initial phosphorylation by cyclin D-CDK4/6 followed by cyclin E-CDK2 phosphorylation (6). Specificity of different CDK/cyclin complexes has been observed in vitro (6-8) and cyclin D1 is required for Ser780 phosphorylation in vivo (9).

  1. Sherr, C.J. (1996) Science 274, 1672-7.
  2. Nevins, J.R. (1992) Science 258, 424-9.
  3. Welch, P.J. and Wang, J.Y. (1993) Cell 75, 779-90.
  4. Hu, Q.J. et al. (1990) EMBO J 9, 1147-55.
  5. Knudsen, E.S. and Wang, J.Y. (1997) Mol Cell Biol 17, 5771-83.
  6. Lundberg, A.S. and Weinberg, R.A. (1998) Mol Cell Biol 18, 753-61.
  7. Connell-Crowley, L. et al. (1997) Mol Biol Cell 8, 287-301.
  8. Kitagawa, M. et al. (1996) EMBO J 15, 7060-9.
  9. Geng, Y. et al. (2001) Proc Natl Acad Sci USA 98, 194-9.

Application References

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Protocols

Companion Products

For Research Use Only. Not For Use In Diagnostic Procedures.

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