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REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous 92 Mouse IgG1
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Flow Cytometry

Flow cytometric analysis of HeLa cells using β-Catenin (L54E2) Mouse mAb (Biotinylated) (blue) compared to Mouse (MOPC-21) mAb IgG1 Isotype Control (Biotinylated) #4097 (red).

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Flow Cytometry

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.
  5. Streptavidin conjugated.

B. 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 10 min at 37°C.
  4. Chill tubes on ice for 1 min.
  5. For extracellular staining with antibodies that do not require permeabilization, proceed to immunostaining (Section D) or store cells in PBS with 0.1% sodium azide at 4°C; for intracellular staining, proceed to permeabilization (Section C).

C. Permeabilization

NOTE: This step is critical for many CST antibodies.

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

D. Immunostaining

NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemocytometer or alternative method.

  1. Aliquot 0.5–1 x 106 cells into each assay tube (by volume).
  2. Add 2–3 ml incubation buffer to each tube and wash by centrifugation. Repeat.
  3. Resuspend cells in 100 µl of diluted, biontinylated primary antibody (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature.
  5. Wash by centrifugation in 2–3 ml incubation buffer.
  6. Resuspend cells in Streptavidin, diluted in incubation buffer at the recommended dilution.
  7. Incubate for 30 min at room temperature.
  8. Wash by centrifugation in 2–3 ml incubation buffer.
  9. Resuspend cells in 0.5 ml 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 30 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised September 2013

protocol id: 160

Product Usage Information

Application Dilutions
Flow Cytometry 1:400

Storage: Supplied in 136 mM NaCl, 2.6 mM KCI, 12 mM sodium phosphate (pH 7.4) dibasic, 2 mg/ml BSA, and 50% glycerol. Store at –20°C. Do not aliquot the antibodies.

Specificity / Sensitivity

β-Catenin (L54E2) Mouse mAb (Biotinylated) detects endogenous levels of total β-catenin protein.


Species Reactivity: Human
Species predicted to react based on 100% sequence homology: Mouse, Rat, Pig

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the carboxy terminus of human β-catenin protein.

Product Description

This Cell Signaling Technology antibody is conjugated to biotin under optimal conditions. The unconjugated β-Catenin (L54E2) Mouse mAb (IF Preferred) #2677 reacts with human β-catenin protein.


β-Catenin is a key downstream effector in the Wnt signaling pathway (1). It is implicated in two major biological processes in vertebrates: early embryonic development (2) and tumorigenesis (3). CK1 phosphorylates β-catenin at Ser45. This phosphorylation event primes β-catenin for subsequent phosphorylation by GSK-3β (4-6). GSK-3β destabilizes β-catenin by phosphorylating it at Ser33, Ser37, and Thr41 (7). Mutations at these sites result in the stabilization of β-catenin protein levels and have been found in many tumor cell lines (8).


1.  Cadigan, K.M. and Nusse, R. (1997) Genes Dev 11, 3286-305.

2.  Wodarz, A. and Nusse, R. (1998) Annu Rev Cell Dev Biol 14, 59-88.

3.  Polakis, P. (1999) Curr Opin Genet Dev 9, 15-21.

4.  Amit, S. et al. (2002) Genes Dev 16, 1066-76.

5.  Liu, C. et al. (2002) Cell 108, 837-47.

6.  Yanagawa, S. et al. (2002) EMBO J 21, 1733-42.

7.  Yost, C. et al. (1996) Genes Dev 10, 1443-54.

8.  Morin, P.J. et al. (1997) Science 275, 1787-90.


Entrez-Gene Id 1499
Swiss-Prot Acc. P35222


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