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11880
Phospho-HS1 (Tyr397) (D12C1) XP® Rabbit mAb (PE Conjugate)
Antibody Conjugates

Phospho-HS1 (Tyr397) (D12C1) XP® Rabbit mAb (PE Conjugate) #11880

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Flow cytometric analysis of Ramos cells, untreated (blue) or treated with anti-IgM and hydrogen peroxide (green), using Phospho-HS1 (Tyr397) (D12C1) XP® Rabbit mAb (PE Conjugate).

To Purchase # 11880S
Product # Size Price
11880S
100 µl  (50 tests) $ 364

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 cytometry analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-HS1 (Tyr397) (D12C1) XP® Rabbit mAb #8714.

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

Phospho-HS1 (Tyr397) (D12C1) XP® Rabbit mAb (PE Conjugate) recognizes endogenous levels of HS1 protein only when phosphorylated at Tyr397.

Species Reactivity:

Human

Species predicted to react based on 100% sequence homology:

Mouse, Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr405 of mouse HS1 protein. This site corresponds to Tyr397 of human HS1 protein.

Background

HS1 (HCLS1, LckBP1, p75) is a protein kinase substrate that is expressed only in tissues and cells of hematopoietic origin (1,2). HS1 contains four cortactin repeats and a single SH3 domain (2). This intracellular protein is phosphorylated following immune receptor activation, which promotes recruitment of HS1 to the immune synapse (3-5). Phosphorylation of HS1 is required to regulate actin dynamics and provide docking sites for many other signaling molecules, such as Vav1 and PLCγ1 (6). HS1 also plays an important role in platelet activation (7).

HS1 is rapidly phosphorylated at Tyr397 by Syk and/or Lyn kinases following immune receptor stimulation and thrombin-mediated platelet stimulation. This phosphorylation is an important step in cytoskeletal rearrangement and signaling complex formation (6-10).

  1. Kitamura, D. et al. (1989) Nucleic Acids Res 17, 9367-79.
  2. Kitamura, D. et al. (1995) Biochem Biophys Res Commun 208, 1137-46.
  3. Suzuki, H. et al. (1997) J Immunol 159, 5881-8.
  4. Hata, D. et al. (1994) Immunol Lett 40, 65-71.
  5. Yamanashi, Y. et al. (1993) Proc Natl Acad Sci USA 90, 3631-5.
  6. Gomez, T.S. et al. (2006) Immunity 24, 741-52.
  7. Kahner, B.N. et al. (2007) Blood 110, 2449-56.
  8. Yamanashi, Y. et al. (1997) J Exp Med 185, 1387-92.
  9. Hao, J.J. et al. (2004) J Biol Chem 279, 33413-20.
  10. Brunati, A.M. et al. (2005) J Biol Chem 280, 21029-35.

Pathways & Proteins

Explore pathways + proteins related to this product.

For Research Use Only. Not For Use In Diagnostic Procedures.

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