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

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

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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).

To Purchase # 80994S
Product # Size Price
80994S
100 µl  (50 tests) N/A

Supporting Data

REACTIVITY H
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 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.

Product Usage Information

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

Species Reactivity:

Human

Source / Purification

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

Background

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.

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.
XP is a registered trademark of Cell Signaling Technology, Inc.

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