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65356
Coronin 1A (D6K5B) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate)
Antibody Conjugates

Coronin 1A (D6K5B) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) #65356

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Flow cytometric analysis of MDA-MB-231 cells (blue) and Jurkat cells (green) using Coronin 1A (D6K5B) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 647 Conjugate) #2985 (dashed lines).

To Purchase # 65356S
Product # Size Price
65356S
100 µl  (50 tests) $ 348

Supporting Data

REACTIVITY H M
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 Alexa Fluor® 647 fluorescent dye 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 Coronin 1A (D6K5B) XP® Rabbit mAb #92904.

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

Coronin 1A (D6K5B) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) recognizes endogenous levels of total coronin 1A protein. Based on the sequence of the immunogenic peptide, this antibody is not expected to cross-react with other coronin protein family members.

Species Reactivity:

Human, Mouse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val200 of human coronin 1A protein.

Background

The coronin family of actin-binding proteins regulates a variety of cellular functions, including migration, phagocytosis, and cytokinesis. Coronin 1A is highly expressed in lymphocytes, and is required for appropriate immune regulation in mice and humans. Researchers are investigating coronin 1A as a potential therapeutic target for autoimmune diseases and lymphoid cancers (1,2). Coronin 1A affects bone resorption through its regulation of lysosome fusion and secretion of cathepsin K in osteoclasts (3). In the nervous system, coronin 1A has been shown to regulate GPCR signaling and neurite outgrowth (4,5).

  1. Punwani, D. et al. (2015) J Clin Immunol 35, 100-7.
  2. Siegmund, K. et al. (2016) J Biol Chem 291, 22086-22092.
  3. Ohmae, S. et al. (2017) Sci Rep 7, 41710.
  4. Martorella, M. et al. (2017) Vitam Horm 104, 113-131.
  5. Terzi, Y.K. et al. (2014) Neurosci Lett 582, 38-42.

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.
This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is conditioned on the buyer using the purchased product solely in research conducted by the buyer, excluding contract research or any fee for service research, and the buyer must not (1) use this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; (b) testing, analysis or screening services, or information in return for compensation on a per-test basis; or (c) manufacturing or quality assurance or quality control, and/or (2) sell or transfer this product or its components for resale, whether or not resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@lifetech.com.