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11917S 100 µl (50 tests) $329.00.0
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REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R Mk Endogenous Rabbit IgG
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Flow Cytometry

Flow cytometric analysis of BT-549 (blue) and Jurkat (green) cells using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb (PE Conjugate).

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Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. 100% methanol.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 15 min at room temperature.
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

  1. Aliquot desired number of cells into tubes or wells.
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

E. Optional DNA Dye

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 5 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised June 2017

Protocol Id: 407

Product Usage Information

Application Dilutions
Flow Cytometry 1:50

Storage: Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibodies. Protect from light. Do not freeze.

Specificity / Sensitivity

Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb (PE Conjugate) recognizes endogenous levels of Rb protein only when phosphorylated at Ser807, Ser811, or at both sites. This antibody does not cross-react with Rb phosphorylated at Ser608.


Species Reactivity: Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser807/811 of human Rb protein.

Product Description

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb #8516.


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.


Entrez-Gene Id 5925
Swiss-Prot Acc. P06400


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.

11917
Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb (PE Conjugate)