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REACTIVITY SENSITIVITY MW (kDa) Isotype
M R Endogenous Rabbit 
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Flow Cytometry

Flow cytometric analysis of Raw 264.7 cells using IKKε (D61F9) XP® Rabbit mAb (PE Conjugate) (blue) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (red).

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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

IKKε (D61F9) XP® Rabbit mAb (PE Conjugate) recognizes endogenous levels of total IKKε protein.


Species Reactivity: Mouse, Rat

Source / Purification

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

Product Description

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in mouse cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated IKKε (D61F9) XP® Rabbit mAb #3416.


The NF-κB/Rel transcription factors are present in the cytosol in an inactive state, complexed with the inhibitory IκB proteins (1-3). Most agents that activate NF-κB do so through a common pathway based on phosphorylation-induced, proteasome-mediated degradation of IκB (3-7). The key regulatory step in this pathway involves activation of a high molecular weight IκB kinase (IKK) complex whose catalysis is generally carried out by three tightly associated IKK subunits. IKKα and IKKβ serve as the catalytic subunits of the kinase and IKKγ serves as the regulatory subunit (8,9). Activation of IKK depends upon phosphorylation at Ser177 and Ser181 in the activation loop of IKKβ (Ser176 and Ser180 in IKKα), which causes conformational changes, resulting in kinase activation (10-13).


1.  Baeuerle, P.A. and Baltimore, D. (1988) Science 242, 540-6.

2.  Beg, A.A. and Baldwin, A.S. (1993) Genes Dev 7, 2064-70.

3.  Finco, T.S. et al. (1994) Proc Natl Acad Sci USA 91, 11884-8.

4.  Brown, K. et al. (1995) Science 267, 1485-8.

5.  Brockman, J.A. et al. (1995) Mol Cell Biol 15, 2809-18.

6.  Traenckner, E.B. et al. (1995) EMBO J 14, 2876-83.

7.  Chen, Z.J. et al. (1996) Cell 84, 853-62.

8.  Zandi, E. et al. (1997) Cell 91, 243-52.

9.  Karin, M. (1999) Oncogene 18, 6867-74.

10.  DiDonato, J.A. et al. (1997) Nature 388, 548-54.

11.  Mercurio, F. et al. (1997) Science 278, 860-6.

12.  Johnson, L.N. et al. (1996) Cell 85, 149-58.

13.  Delhase, M. et al. (1999) Science 284, 309-13.


Entrez-Gene Id 9641
Swiss-Prot Acc. Q14164


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.

85033
IKKε (D61F9) XP® Rabbit mAb (PE Conjugate)