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

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

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Flow cytometric analysis of HL-60 cells (blue) and K-562 cells (green) using Annexin A2 (D11G2) Rabbit mAb (PE Conjugate).

To Purchase # 15161S
Product # Size Price
15161S
100 µl  (50 tests) $ 305

Supporting Data

REACTIVITY H M R Mk B Pg
SENSITIVITY Endogenous
MW (kDa)
Isotype Rabbit IgG

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

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.

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.

Protocol

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Flow Cytometry, Methanol Permeabilization Protocol for Directly Conjugated Antibodies

A. Solutions and Reagents

All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
  2. 4% Formaldehyde, Methanol-Free (#47746)
  3. 100% Methanol (#13604): Chill before use
  4. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com/flowdyes for a listing of cellular dyes validated for use in flow cytometry.

B. Fixation

NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.

  1. Pellet cells by centrifugation and remove supernatant.
  2. Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
  3. Fix for 15 min at room temperature (20-25°C).
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS. Proceed to Permeabilization step.
    1. Alternatively, cells may be stored overnight at 4°C in 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. Permeabilize for a minimum of 10 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
  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 primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
  6. Resuspend cells in 200-500 µl of 1X PBS and analyze on flow cytometer.

posted July 2009

revised August 2019

Protocol Id: 407

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.

Background

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.

Pathways & Proteins

Explore pathways + proteins related to this product.

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.