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23353S 100 µl (50 tests) $329.00.0
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REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous Rabbit IgG
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Flow Cytometry

Flow cytometric analysis of MDA-MB-231 cells (blue) and T47D cells (green) using Progesterone Receptor A/B (D8Q2J) 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

Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb (PE Conjugate) recognizes endogenous levels of total progesterone receptor A and B proteins. This antibody does not cross-react with either the glucocorticoid receptor or the mineralocorticoid receptor.


Species Reactivity: Human
Species predicted to react based on 100% sequence homology: Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Tyr541 of human progesterone receptor 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 Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb #8757.


Human progesterone receptor (PR) is expressed as two forms: the full length PR-B and the short form PR-A. PR-A lacks the first 164 amino acid residues of PR-B (1,2). Both PR-A and PR-B are ligand activated, but differ in their relative ability to activate target gene transcription (3,4). The activity of PR is regulated by phosphorylation; at least seven serine residues are phosphorylated in its amino-terminal domain. Three sites (Ser81, Ser102, and Ser162) are unique to full length PR-B, while other sites (Ser190, Ser294, Ser345, and Ser400) are shared by both isoforms (5). Phosphorylation of PR-B at Ser190 (equivalent to Ser26 of PR-A) is catalyzed by CDK2 (6). Mutation of Ser190 results in decreased activity of PR (7), suggesting that the phosphorylation at Ser190 may be critical to its biological function.


1.  Evans, R.M. (1988) Science 240, 889-95.

2.  Kastner, P. et al. (1990) EMBO J 9, 1603-14.

3.  Giangrande, P.H. et al. (2000) Mol Cell Biol 20, 3102-15.

4.  Wen, D.X. et al. (1994) Mol Cell Biol 14, 8356-64.

5.  Clemm, D.L. et al. (2000) Mol. Endocrinol. 14, 52-65.

6.  Zhang, Y. et al. (1997) Mol. Endocrinol. 11, 823-832.

7.  Takimoto, G.S. et al. (1996) J. Biol. Chem. 271, 13308-13316.


Entrez-Gene Id 5241
Swiss-Prot Acc. P06401


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.

23353
Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAb (PE Conjugate)