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15161S 100 µl (50 tests) $299.00.0
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REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R Mk B Pg Endogenous Rabbit IgG
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Flow Cytometry

Flow cytometric analysis of HL-60 cells (blue) and K-562 cells (green) using Annexin A2 (D11G2) 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

Annexin A2 (D11G2) Rabbit mAb (PE Conjugate) recognizes endogenous levels of total annexin A2 protein. This antibody is not known or predicted to cross-react with other annexin family members.


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

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Phe307 of human annexin A2 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. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated Annexin A2 (D11G2) Rabbit mAb #8235.


Annexin A2 (ANXA2), also known as lipocortin II or calpactin-1 heavy chain, is a 36 kDa member of the annexin superfamily that binds phospholipids and other proteins in a calcium-dependent manner via annexin repeats (1). Annexin A2 contains four such repeats through which it mediates protein-protein and protein-lipid interactions (1-4). It forms a constitutive heterotetramer with S100A10, acting as a bridge between the actin cytoskeleton, plasma membrane, and endocytotic vesicle machinery (5-7). Originally identified as a protein inhibitor of phospholipase A2, annexin A2 has subsequently been shown to interact with an array of protein and non-protein partners, including F-actin, spectrin, SNARE complexes, RNA, and virus particles (4,6,8,9). Annexin A2 has also been shown to have receptor-like activity and is detected on the surface of macrophages and vascular endothelial cells where it mediates macrophage activation and Factor Xa signaling, respectively (10-13). Upregulation of annexin A2 at the cell surface is thought to be modulated by phosphorylation at Tyr23 by Src (14-18). Interestingly, phosphorylation at Tyr23 has recently been shown to be required for cell surface expression of annexin A2 where it mediates motility, invasiveness, and overall metastatic potential of certain pancreatic cancer cells (19,20). Annexin A2 has also been shown to be heavily phosphorylated on serine residues in response to PKC activation via a pleiotropic mechanism (21-23). For a complete list of curated phosphorylation sites on annexin A2, please see PhosphoSitePlus® at www.phosphosite.org.


1.  Barton, G.J. et al. (1991) Eur J Biochem 198, 749-60.

2.  Gerke, V. and Weber, K. (1985) EMBO J 4, 2917-20.

3.  Glenney, J.R. and Tack, B.F. (1985) Proc Natl Acad Sci USA 82, 7884-8.

4.  Gerke, V. and Weber, K. (1984) EMBO J 3, 227-33.

5.  Illien, F. et al. (2010) Biochim Biophys Acta 1798, 1790-6.

6.  Umbrecht-Jenck, E. et al. (2010) Traffic 11, 958-71.

7.  Jung, M.J. et al. (2010) Exp Cell Res 316, 1234-40.

8.  Filipenko, N.R. et al. (2004) J Biol Chem 279, 8723-31.

9.  Wright, J.F. et al. (1994) Biochem Biophys Res Commun 198, 983-9.

10.  Bhattacharjee, G. et al. (2008) Circ Res 102, 457-64.

11.  Pizzo, S.V. (2008) Circ Res 102, 389-91.

12.  Swisher, J.F. et al. (2007) J Leukoc Biol 82, 1174-84.

13.  Deora, A.B. et al. (2004) J Biol Chem 279, 43411-8.

14.  Huang, K.S. et al. (1986) Cell 46, 191-9.

15.  Erikson, E. et al. (1984) Mol Cell Biol 4, 77-85.

16.  Glenney, J.R. (1985) FEBS Lett 192, 79-82.

17.  Morel, E. and Gruenberg, J. (2009) J Biol Chem 284, 1604-11.

18.  de Graauw, M. et al. (2008) Mol Cell Biol 28, 1029-40.

19.  Nedjadi, T. et al. (2009) Br J Cancer 101, 1145-54.

20.  Zheng, L. et al. (2011) PLoS One 6, e19390.

21.  Gould, K.L. et al. (1986) Mol Cell Biol 6, 2738-44.

22.  Luo, W. et al. (2008) Mol Carcinog 47, 934-46.

23.  He, K.L. et al. (2011) J Biol Chem 286, 15428-39.


Entrez-Gene Id 302
Swiss-Prot Acc. P07355


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

15161
Annexin A2 (D11G2) Rabbit mAb (PE Conjugate)