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87593S 100 µl (50 tests) $299.00.0


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H M R Endogenous Rabbit IgG

Flow Cytometry

Flow cytometric analysis of THP-1 cells, untreated (green) or treated with TPA #4174 (80 nM, 16 hr; red), using Phospho-SAMHD1 (Thr592) (D7O2M) Rabbit mAb (PE Conjugate) (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed lines).

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Flow Cytometry

Flow cytometric analysis of Jurkat cells (blue) and THP-1 cells (green) using Phospho-SAMHD1 (Thr592) (D7O2M) Rabbit mAb (PE Conjugate) (solid lines) or a 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

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-SAMHD1 (Thr592) (D7O2M) Rabbit mAb (PE Conjugate) recognizes endogenous levels of SAMDH1 protein only when phosphorylated at Thr592.

Species Reactivity: Human, Mouse, Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Thr592 of human SAMHD1 protein.

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 Phospho-SAMHD1 (Thr592) (D7O2M) Rabbit mAb #89930.

SAM domain and HD domain-containing protein 1 (SAMHD1) is a negative regulator of the cell-intrinsic innate immune response (1). Research studies have identified mutations in SAMHD1 as a cause of Aicardi-Goutieres syndrome, an autoimmune disease characterized by elevated production of interferon-α and symptoms resembling congenital viral infection (1). SAMHD1 was identified as the restriction factor that renders most myeloid cells refractory to human immunodeficiency virus (HIV) infection (2-4). Expression of the viral protein Vpx in refractory cells targets SAMHD1 for ubiquitin-mediated degradation and relieves HIV restriction. SAMHD1 prevents autoimmunity and HIV infection by hydrolyzing intracellular deoxynucleoside triphosphates (dNTPs), thereby limiting inappropriate immune activation by self nucleic acid and inhibiting reverse transcription of the HIV genome (4-6).

1.  Rice, G.I. et al. (2009) Nat Genet 41, 829-32.

2.  Laguette, N. et al. (2011) Nature 474, 654-7.

3.  Hrecka, K. et al. (2011) Nature 474, 658-61.

4.  Powell, R.D. et al. (2011) J Biol Chem 286, 43596-600.

5.  Goldstone, D.C. et al. (2011) Nature 480, 379-82.

6.  Lahouassa, H. et al. (2012) Nat Immunol 13, 223-8.

Entrez-Gene Id 25939
Swiss-Prot Acc. Q9Y3Z3

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.

Phospho-SAMHD1 (Thr592) (D7O2M) Rabbit mAb (PE Conjugate)