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PhosphoSitePlus® Resource

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REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous Rabbit IgG
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Flow Cytometry

Flow cytometric analysis of Ramos cells, untreated (blue) or IgM-treated (green), using Phospho-HS1 (Tyr397) (D12C1) XP® Rabbit mAb (Pacific Blue™ Conjugate).

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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

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 antibody. Protect from light. Do not freeze.

Specificity / Sensitivity

Phospho-HS1 (Tyr397) (D12C1) XP® Rabbit mAb (Pacific Blue™ 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.

Product Description

This Cell Signaling Technology antibody is conjugated to Pacific Blue™ fluorescent dye 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.


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).


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.


Entrez-Gene Id 3059
Swiss-Prot Acc. P14317


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.
Pacific Blue is a trademark of Molecular Probes, Inc.

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Phospho-HS1 (Tyr397) (D12C1) XP® Rabbit mAb (Pacific Blue™ Conjugate)