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56159S 100 µl (50 tests) $299.00.0
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REACTIVITY SENSITIVITY MW (kDa) Isotype
H M Endogenous Rabbit IgG
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Flow Cytometry

Flow cytometric analysis of HeLa (blue) and NTERA-2 (green) cells using Oct-4A (C30A3) Rabbit mAb (PE Conjugate).

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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 antibodies. Protect from light. Do not freeze.

Specificity / Sensitivity

Oct-4A (C30A3) Rabbit mAb (PE Conjugate) recognizes endogenous levels of total Oct-4A protein.


Species Reactivity: Human, Mouse

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the amino terminus of human Oct-4A.

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 Oct-4A (C30A3) Rabbit mAb #2840.


Oct-4 (POU5F1) is a transcription factor highly expressed in undifferentiated embryonic stem cells and embryonic germ cells (1). A network of key factors that includes Oct-4, Nanog, and Sox2 is necessary for the maintenance of pluripotent potential, and downregulation of Oct-4 has been shown to trigger cell differentiation (2,3). Research studies have demonstrated that Oct-4 is a useful germ cell tumor marker (4). Oct-4 exists as two splice variants, Oct-4A and Oct-4B (5). Recent studies have suggested that the Oct-4A isoform has the ability to confer and sustain pluripotency, while Oct-4B may exist in some somatic, non-pluripotent cells (6,7).


1.  Looijenga, L.H. et al. (2003) Cancer Res 63, 2244-50.

2.  Pesce, M. and Schöler, H.R. (2001) Stem Cells 19, 271-8.

3.  Pan, G. and Thomson, J.A. (2007) Cell Res 17, 42-9.

4.  Cheng, L. et al. (2007) J Pathol 211, 1-9.

5.  Takeda, J. et al. (1992) Nucleic Acids Res 20, 4613-20.

6.  Cauffman, G. et al. (2006) Stem Cells 24, 2685-91.

7.  Lee, J. et al. (2006) J Biol Chem 281, 33554-65.


Entrez-Gene Id 5460
Swiss-Prot Acc. Q01860


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 5,675,063.

56159
Oct-4A (C30A3) Rabbit mAb (PE Conjugate)