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50230
BrdU (Bu20a) Mouse mAb (PE Conjugate)
Antibody Conjugates
Monoclonal Antibody

BrdU (Bu20a) Mouse mAb (PE Conjugate) #50230

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Flow Cytometry Image 1: BrdU (Bu20a) Mouse mAb (PE Conjugate)

Flow cytometric analysis of Jurkat cells incorporated with BrdU (30 min), using BrdU (Bu20a) Mouse mAb (PE conjugate) versus DRAQ5® (DNA content).

To Purchase # 50230S
Product # Size Price
50230S
100 µl  (50 tests) N/A

Supporting Data

REACTIVITY All
SENSITIVITY
MW (kDa)
Source/Isotype Mouse IgG1

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
  • 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 cytometry analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated BrdU (Bu20a) Mouse mAb #5292.

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

A. Solutions and Reagents

  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 distilled water (dH2O). Adjust the pH to 7.4 with HCl and the volume to 1 liter. Store at room temperature.
  2. BrdU (5-Bromo-2′-deoxyuridine) EMD biosciences (Cat. #203806)
  3. Ethanol, anhydrous denatured, histological grade, 100% and 95%
  4. 1.5 M Hydrochloric acid
  5. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) in 100 mL 1X PBS. Store at 4°C.
  6. Recommended Anti-Mouse secondary antibodies::

B. BrdU Incorporation and Specimen Preparation

  1. Add BrdU to fresh, warm media for a final concentration of 0.03 mg/mL. Set aside.
  2. Collect ~50 million cells in tube by centrifugation and aspirate supernatant.
  3. Add 2 ml of BrdU-containing media to cell pellet, vortex, and incubate at 37°C for 30 minutes.
  4. Pellet cells by centrifugation and aspirate media.
  5. Add 2 ml of cold, 70% ethanol to cell pellet and mix.
  6. Allow cells to fix for 5 minutes at room temperature.
  7. Add 2–3 ml PBS and rinse by centrifugation three times.
  8. Add 1.5 M HCL and incubate for 30 minutes at room temperature.
  9. Add 2–3 ml PBS and rinse by centrifugation two times.
  10. Proceed with Immunostaining section C.

C. Immunostaining

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

  1. Aliquot 0.5–1 x 106 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 unconjugated primary antibody at the appropriate dilution to the assay tubes (see antibody data sheet 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 fluorochrome-conjugated secondary antibody diluted in Incubation Buffer at the recommended dilution.
  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 D1.

D. Optional DNA Stain

  1. Resuspend cells in 0.5 ml of DNA dye (eg. DRAQ5® #4084).
  2. Incubate for at least 5 mins at room temperature.
  3. Analyze cells in DNA stain on flow cytometer.

posted December 2009

Protocol Id: 30

Specificity / Sensitivity

BrdU (Bu20a) Mouse mAb (PE Conjugate) detects BrdU when incorporated into single stranded DNA. DNA must be denatured for the epitope to be exposed and recognized by the antibody.

Species Reactivity:

All Species Expected

Source / Purification

Monoclonal antibody is produced by immunizing animals with BrdU conjugated to BSA.

Background

Halogenated nucleotides such as the pyrimidine analog bromodeoxyuridine (BrdU) are useful for labeling nascent DNA in living cells and tissues. BrdU becomes incorporated into replicating DNA in place of thymidine and subsequent immunodetection of BrdU using specific monoclonal antibodies allows labeling of cells in S phase of the cell cycle. After pulse-labeling cells or tissues with bromodeoxyuridine, BrdU (Bu20a) Mouse mAb can be used to detect BrdU incorporated into single stranded DNA. Please see our detailed protocol for information regarding the labeling procedure and denaturation of double stranded DNA for various immunodetection applications (1-4).

  1. Darzynkiewicz, Z. and Juan, G. (2001) Curr Protoc Cytom Chapter 7, Unit 7.7.
  2. Leif, R.C. et al. (2004) Cytometry A 58, 45-52.
  3. Staszkiewicz, J. et al. (2009) Biochem Biophys Res Commun 378, 539-44.
  4. Rothaeusler, K. and Baumgarth, N. (2007) Curr Protoc Cytom Chapter 7, Unit7.31.

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.
DRAQ5 is a registered trademark of Biostatus Limited.
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