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61446
PCNA (D3H8P) XP® Rabbit mAb (PE Conjugate)
Antibody Conjugates
Monoclonal Antibody

PCNA (D3H8P) XP® Rabbit mAb (PE Conjugate) #61446

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Flow Cytometry Image 1: PCNA (D3H8P) XP® Rabbit mAb (PE Conjugate)

Flow cytometric analysis of Jurkat cells using PCNA (D3H8P) XP® Rabbit mAb (PE Conjugate) and DRAQ5® #4084 to measure DNA content.

To Purchase # 61446S
Product # Size Price
61446S
100 µl  (50 tests) $ 368

Supporting Data

REACTIVITY H M R Mk
SENSITIVITY Endogenous
MW (kDa)
Source/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
  • Vir-Virus
  • 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 cytometric analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated PCNA (D3H8P) XP® Rabbit mAb #13110.

Product Usage Information

Application Dilution
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.

Protocol

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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

Specificity / Sensitivity

PCNA (D3H8P) XP® Rabbit mAb (PE Conjugate) recognizes endogenous levels of total PCNA protein.

Species Reactivity:

Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human PCNA protein.

Background

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.

Pathways & Proteins

Explore pathways + proteins related to this product.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST's products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST's Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

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.
DRAQ5 is a registered trademark of Biostatus Limited.