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

Flow cytometric analysis of HeLa cells using PIAS1 (D33A7) XP® Rabbit mAb (PE Conjugate) (blue) compared to Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (red).

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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

PIAS1 (D33A7) XP® Rabbit mAb (PE Conjugate) recognizes endogenous levels of total PIAS1 protein.


Species Reactivity: Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser550 of human PIAS1 protein.

Product Description

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated PIAS1 (D33A7) XP® Rabbit mAb #3550.


The protein inhibitor of activated Stat (PIAS) proteins, which include PIAS1, PIAS3, PIASx, and PIASy, were originally characterized based on their interaction with the Stat family of transcription factors (1,2). PIAS1, PIAS3, and PIASx interact with and repress Stat1, Stat3, and Stat4, respectively (1-3). Deletion of PIAS1 leads to inhibition of interferon-inducible genes and increased protection against infection (4). The PIAS family contains a conserved RING domain that has been linked to a function as a small ubiquitin-related modifier (SUMO) ligase, coupling the SUMO conjugating enzyme Ubc9 with its substrate proteins (5,6). Numerous studies have now shown that PIAS family members can regulate the activity of transcription factors through distinct mechanisms, including NF-κB (7,8), c-Jun, p53 (5,9), Oct-4 (10), and Smads (11,12). The activity of PIAS1 is regulated by both phosphorylation and arginine methylation. Inflammatory stimuli can induce IKK-mediated phosphorylation of PIAS1 at Ser90, which is required for its activity (13). In addition, PRMT1 induces arginine methylation of PIAS1 at Arg303 following interferon treatment and is associated with its repressive activity on Stat1 (14).


1.  Liu, B. et al. (1998) Proc Natl Acad Sci USA 95, 10626-31.

2.  Chung, C.D. et al. (1997) Science 278, 1803-5.

3.  Arora, T. et al. (2003) J Biol Chem 278, 21327-30.

4.  Liu, B. et al. (2004) Nat Immunol 5, 891-8.

5.  Schmidt, D. and Müller, S. (2002) Proc Natl Acad Sci USA 99, 2872-7.

6.  Kotaja, N. et al. (2002) Mol Cell Biol 22, 5222-34.

7.  Liu, B. et al. (2005) Mol Cell Biol 25, 1113-23.

8.  Tahk, S. et al. (2007) Proc Natl Acad Sci USA 104, 11643-8.

9.  Bischof, O. et al. (2006) Mol Cell 22, 783-94.

10.  Tolkunova, E. et al. (2007) J Mol Biol 374, 1200-12.

11.  Long, J. et al. (2004) Proc Natl Acad Sci USA 101, 99-104.

12.  Murdoch, R.N. and Edwards, T. (1992) Biochem Int 28, 1029-37.

13.  Liu, B. et al. (2007) Cell 129, 903-14.

14.  Weber, S. et al. (2009) Genes Dev 23, 118-32.


Entrez-Gene Id 8554
Swiss-Prot Acc. O75925


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.

92725
PIAS1 (D33A7) XP® Rabbit mAb (PE Conjugate)