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Annexin A1 (D5V2T) XP® Rabbit mAb (PE Conjugate)
Antibody Conjugates

Annexin A1 (D5V2T) XP® Rabbit mAb (PE Conjugate) #80994


H Endogenous Rabbit IgG
Flow Cytometry

Flow cytometric analysis of 293T cells (blue) or ACHN cells (green) using Annexin A1 (D5V2T) XP® Rabbit mAb (PE Conjugate) (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed lines).

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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

Application Dilutions
Flow Cytometry 1:50

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 antibody. Protect from light. Do not freeze.

Annexin A1 (D5V2T) XP® Rabbit mAb (PE Conjugate) recognizes endogenous levels of total annexin A1 protein.

Species Reactivity:


Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human annexin A1 protein.

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometric analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated Annexin A1 (D5V2T) XP® Rabbit mAb #32934.

The annexin superfamily consists of 13 calcium or calcium and phospholipid binding proteins with high biological and structural homology (1). Annexin-1 (ANXA1) is the first characterized member of the annexin family of proteins and is able to bind to cellular membranes in a calcium-dependent manner, promoting membrane fusion and endocytosis (2-4). Annexin A1 has anti-inflammatory properties and inhibits phospholipase A2 activity (5,6). Annexin A1 can accumulate on internalized vesicles after EGF-stimulated endocytosis and may be required for a late stage in inward vesiculation (7). Phosphorylation by PKC, EGFR, and Chak1 results in inhibition of annexin A1 function (8-10). Annexin A1 has also been identified as one of the 'eat-me' signals on apoptotic cells that are to be recognized and ingested by phagocytes (11). Annexin A1, as an endogenous anti-inflammatory mediator, has roles in many diverse cellular functions, such as membrane aggregation, inflammation, phagocytosis, proliferation, apoptosis, and tumorigenesis and cancer development (12-14).

  1. Raynal, P. and Pollard, H.B. (1994) Biochim Biophys Acta 1197, 63-93.
  2. Blackwell, G.J. et al. (1980) Nature 287, 147-9.
  3. Rothhut, B. et al. (1983) Biochem Biophys Res Commun 117, 878-84.
  4. Hirata, F. et al. (1981) Proc Natl Acad Sci USA 78, 3190-4.
  5. Kim, K.M. et al. (1994) FEBS Lett 343, 251-5.
  6. Kim, S.W. et al. (2001) J Biol Chem 276, 15712-9.
  7. White, I.J. et al. (2006) EMBO J 25, 1-12.
  8. Varticovski, L. et al. (1988) Biochemistry 27, 3682-90.
  9. Dorovkov, M.V. and Ryazanov, A.G. (2004) J Biol Chem 279, 50643-6.
  10. Wang, W. and Creutz, C.E. (1994) Biochemistry 33, 275-82.
  11. Arur, S. et al. (2003) Dev Cell 4, 587-98.
  12. Perretti, M. and Gavins, F.N. (2003) News Physiol Sci 18, 60-4.
  13. Parente, L. and Solito, E. (2004) Inflamm Res 53, 125-32.
  14. Lim, L.H. and Pervaiz, S. (2007) FASEB J 21, 968-75.
Entrez-Gene Id
Swiss-Prot Acc.
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.

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Product # Size
100 µl (50 tests)

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