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14710S 100 µl (50 tests) $329.00.0
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REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R Hm Mk Endogenous Rabbit IgG
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Flow Cytometry

Flow cytometric analysis of RL-7 cells (blue) and A-204 cells (green) using YAP (D8H1X) 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

YAP (D8H1X) XP® Rabbit mAb (PE Conjugate) recognizes endogenous levels of total YAP protein.


Species Reactivity: Human, Mouse, Rat, Hamster, Monkey
Species predicted to react based on 100% sequence homology: Bovine, Guinea Pig, Horse

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the carboxy terminus of human YAP protein. The epitope corresponds to a region surrounding Pro435 of human YAP isoform 1. This sequence region is 100% conserved among all known isoforms of human YAP 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 YAP (D8H1X) XP® Rabbit mAb #14074.


YAP (Yes-associated protein, YAP65) was identified based on its ability to associate with the SH3 domain of Yes. It also binds to other SH3 domain-containing proteins such as Nck, Crk, Src, and Abl (1). In addition to the SH3 binding motif, YAP contains a PDZ interaction motif, a coiled-coil domain, and WW domains (2-4). While initial studies of YAP all pointed towards a role in anchoring and targeting to specific subcellular compartments, subsequent studies showed that YAP is a transcriptional co-activator by virtue of its WW domain interacting with the PY motif (PPxY) of the transcription factor PEBP2 and other transcription factors (5,6). In its capacity as a transcriptional co-activator, YAP is now widely recognized as a central mediator of the Hippo Pathway, which plays a fundamental and widely conserved role in regulating tissue growth and organ size. Phosphorylation at multiple sites (e.g., Ser109, Ser127) by LATS1/2 kinases promotes YAP translocation from the nucleus to the cytoplasm, where it is sequestered through association with 14-3-3 proteins in an Akt-dependent manner (7-9). These LATS-driven phosphorylation events serve to prime YAP for subsequent phosphorylation by CK1δ/ε in an adjacent phosphodegron, triggering proteosomal degradation of YAP (10).


1.  Sudol, M. (1994) Oncogene 9, 2145-52.

2.  Mohler, P.J. et al. (1999) J Cell Biol 147, 879-90.

3.  Espanel, X. and Sudol, M. (2001) J Biol Chem 276, 14514-23.

4.  Sudol, M. et al. (1995) FEBS Lett 369, 67-71.

5.  Yagi, R. et al. (1999) EMBO J 18, 2551-62.

6.  Basu, S. et al. (2003) Mol Cell 11, 11-23.

7.  Zhao, B. et al. (2010) Genes Dev 24, 862-74.

8.  Zhao, B. et al. (2007) Genes Dev 21, 2747-61.

9.  Yu, F.X. et al. (2012) Cell 150, 780-91.

10.  Zhao, B. et al. (2010) Genes Dev 24, 72-85.


Entrez-Gene Id 10413
Swiss-Prot Acc. P46937


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.

14710
YAP (D8H1X) XP® Rabbit mAb (PE Conjugate)