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59056S 100 µl (50 assays) $299.00.0
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REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R Mk Endogenous Rabbit 
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Flow Cytometry

Flow Cytometric analysis of 293T cells, untreated (blue) or UV-treated (green), using Phospho-SEK1/MKK4(Ser257)(C36C11) 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 antibody. Protect from light. Do not freeze.

Specificity / Sensitivity

Phospho-SEK1/MKK4 (Ser257) (C36C11) Rabbit mAb (PE Conjugate) detects endogenous levels of SEK1/MKK4 phosphorylated at Ser257.


Species Reactivity: Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser257/Thr261 of human SEK1/MKK4.

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 Phospho-SEK1/MKK4 (Ser257) (C36C11) Rabbit mAb #4514.


SAPK/Erk kinase (SEK1), also known as MKK4 or Jun kinase kinase (JNKK), activates the MAP kinase homologues SAPK and JNK in response to various cellular stresses and inflammatory cytokines (1-3). Activation of SEK1 occurs through MEKK phosphorylation of serine and threonine residues at positions 257 and 261, respectively. Like MEK, SEK is a dual-specificity protein kinase that phosphorylates SAPK/JNK at a conserved T*PY* site in its activation loop (4). Phosphorylation by Akt at Ser80 inhibits SEK1 and suppresses stress-activated signal transduction (5).


1.  Davis, R.J. (1994) Trends Biochem. Sci. 19, 470-473.

2.  Sanchez, I. et al. (1994) Nature 372, 794-798.

3.  Yan, M. et al. (1994) Nature 372, 798-800.

4.  Kyriakis, J.M. et al. (1994) Nature 369, 156-160.

5.  Park, H. et al. (2002) J. Biol. Chem. 277, 2573-2578.


Entrez-Gene Id 6416
Swiss-Prot Acc. P45985


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

59056
Phospho-SEK1/MKK4 (Ser257) (C36C11) Rabbit mAb (PE Conjugate)