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Dec 31st Santa Cruz will discontinue a
large number of polyclonal products as
a result of the USDA settlement that was
made public May 19th.

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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 General Protocol

If using whole blood, please follow the Flow Cytometry Whole Blood Protocol.

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

  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 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 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

Flow Cytometry Whole Blood Protocol

If using cell lines, please follow the Flow Cytometry General Protocol.

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. Triton™ X-100: To prepare 50 ml of 0.1% Triton™ X-100 add 50 μl Triton™ X-100 to 50 ml 1 X PBS and mix well.
  4. 50% methanol.
  5. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Preparation of Whole Blood (fixation, lysis, and permeabilization) for Immunostaining

  1. Aliquot 100 μl fresh whole blood per assay tube.
  2. OPTIONAL: Place tubes in rack in 37°C water bath for short-term treatments with ligands, inhibitors, drugs, etc.
  3. Add 65 μl of 10% formaldehyde to each tube.
  4. Vortex briefly and let stand for 15 min at room temperature.
  5. Add 1 ml of 0.1% Triton™ X-100 to each tube.
  6. Vortex and let stand for 30 min at room temperature.
  7. Add 1 ml incubation buffer.
  8. Pellet cells by centrifugation and aspirate supernatant.
  9. Repeat steps 7 and 8.
  10. Resuspend cells in ice-cold 50% methanol in PBS (store methanol solution at -20°C until use).
  11. Incubate at least 10 min on ice.
  12. Proceed with staining or store cells at -20°C in 50% methanol.

C. Staining Using Conjugated Primary Antibodies

NOTE: Account for isotype-matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies.

  1. Add 2–3 ml incubation buffer to each tube and rinse by centrifugation. Repeat.
  2. Add primary antibodies diluted as recommended on datasheet or product webpage in incubation buffer.
  3. Incubate for 1 hr at room temperature.
  4. Wash by centrifugation in 2–3 ml incubation buffer.
  5. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.

Reference: Chow S, Hedley D, Grom P, Magari R, Jacobberger JW, Shankey TV (2005) Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry A 67(1), 4–17.

posted November 2008

revised September 2013

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)