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REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R Mk B Pg Endogenous Mouse IgG2a
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Flow cytometric analysis of Jurkat cells pre-extracted with Triton-X-100 (see protocol) using PCNA (PC10) Mouse mAb (PE Conjugate) and DRAQ5® #4084 (DNA content).

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Flow Cytometry

NOTE: Prepare solutions with Milli-Q or equivalently purified water.

  1. 1X Phosphate Buffered Saline (PBS): Dissolve 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 in 800 ml dH2O. Adjust the pH to 7.4 with HCl and the volume to 1 L. Store at room temperature.
  2. Absolute Methanol
  3. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) in 100 ml 1X PBS. Store at 4°C.
  4. Permeabilization Buffer: 0.5% Triton X-100, 0.2 µg/ml EDTA and 1% BSA in PBS.
  5. Recommended Anti-Mouse secondary antibodies::

A. Isolation of Cells and Fixation

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells briefly in 100 µl PBS.
  3. Add 500 µl of permeabilization buffer. Incubate for 15 minutes on ice.
  4. Add 3 ml of ice-cold 100% methanol and mix on and off for 10 minutes.
  5. Proceed with staining or store cell at -20°C.

B. Immunostaining

NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemocytometer or alternative method.

  1. Aliquot 0.5–1x106 cells into each assay tube (by volume).
  2. Add 2–3 ml Incubation Buffer to each tube and rinse by centrifugation. Repeat.
  3. Resuspend cells in 100 µl Incubation Buffer per assay tube.
  4. Block in Incubation Buffer for 10 minutes at room temperature.
  5. Add the fluorochrome-conjugated primary antibody at the appropriate dilution to the assay tubes (see individual antibody datasheet for the appropriate dilution).
  6. Incubate for 1 hour at room temperature.
  7. Rinse as before in Incubation Buffer by centrifugation.
  8. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.
  9. Incubate for 30 minutes at room temperature.
  10. Rinse as before in Incubation Buffer by centrifugation.
  11. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to step C1.

C. Optional DNA Stain

  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 minutes at room temperature.

Analyze cells in DNA stain on flow cytometer.

posted April 2012

protocol id: 45

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.

Specificity / Sensitivity

PCNA (PC10) Mouse mAb (PE Conjugate) detects endogenous levels of total PCNA protein.


Species Reactivity: Human, Mouse, Rat, Monkey, Bovine, Pig

Source / Purification

Monoclonal antibody is produced by immunizing animals with a recombinant Protein A-PCNA fusion protein obtained from pC2T.

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 PCNA (PC10) Mouse mAb #2586.


Proliferating cell nuclear antigen (PCNA) is a member of the DNA sliding clamp family of proteins that assist in DNA replication (1). Crystal structure data suggests that a PCNA homotrimer ring can encircle and slide along the DNA double helix (2). Multiple proteins involved in DNA replication, DNA repair, and cell cycle control bind to PCNA rather than directly associating with DNA, thus facilitating fast processing of DNA (reviewed in 3). PCNA protein expression is a well-accepted marker of proliferation.


1.  Kelman, Z. and O'Donnell, M. (1995) Nucleic Acids Res 23, 3613-20.

2.  Krishna, T.S. et al. (1994) Cell 79, 1233-43.

3.  Maga, G. and Hubscher, U. (2003) J Cell Sci 116, 3051-60.


Entrez-Gene Id 5111
Swiss-Prot Acc. P12004


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
DRAQ5® is a registered trademark of Biostatus Limited.