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64728
Wee1 (D10D2) Rabbit mAb (PE Conjugate)

Wee1 (D10D2) Rabbit mAb (PE Conjugate) #64728

APPLICATIONS

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

Flow cytometric analysis of Jurkat cells using Wee1 (D10D2) Rabbit mAb (PE Conjugate) and DRAQ5® #4084 to measure DNA content.

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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

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.

Wee1 (D10D2) Rabbit mAb (PE Conjugate) recognizes endogenous levels of total Wee1 protein.

Species Reactivity:

Human, Monkey

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human Wee1 protein.

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 Wee1 (D10D2) Rabbit mAb #13084.

Entry of all eukaryotic cells into mitosis is regulated by activation of cdc2 kinase. The critical regulatory step in activating cdc2 during progression into mitosis appears to be dephosphorylation of Tyr15 and Thr14 (1,2). Phosphorylation at Tyr15 and Thr14 and inhibition of cdc2 is carried out by Wee1 and Myt1 protein kinases, while Tyr15 dephosphorylation and activation of cdc2 is carried out by the cdc25 phosphatase (1,3,4). Hyperphosphorylation and inactivation of Myt1 in mitosis suggests that one or more kinases activated at the G2/M transition negatively regulates Myt1 activity. Kinases shown to phosphorylate Myt1 include cdc2, p90RSK, Akt, and Plk1 (5-8).

  1. Watanabe, N. et al. (1995) EMBO J 14, 1878-91.
  2. Hunter, T. (1995) Cell 80, 225-36.
  3. Galaktionov, K. et al. (1995) Genes Dev 9, 1046-58.
  4. McGowan, C.H. and Russell, P. (1993) EMBO J 12, 75-85.
  5. Booher, R.N. et al. (1997) J Biol Chem 272, 22300-6.
  6. Palmer, A. et al. (1998) EMBO J 17, 5037-47.
  7. Palmer, A. et al. (1998) EMBO J 17, 5037-47.
  8. Nakajima, H. et al. (2003) J Biol Chem 278, 25277-80.
Entrez-Gene Id
7465
Swiss-Prot Acc.
P30291
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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To Purchase # 64728S

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Product # Size Price
64728S
100 µl (50 tests) $ 299.0

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