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

Flow cytometric analysis of Raji cells, untreated (blue) or treated with TPA #4174 (200 nM, 30 mins) (green), using Phospho-RelB (Ser552) (D41B9) XP® 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

Phospho-RelB (Ser552) (D41B9) XP® Rabbit mAb (PE Conjugate) detects endogenous levels of RelB protein only when phosphorylated at Ser552.


Species Reactivity: Human, Mouse
Species predicted to react based on 100% sequence homology: Rat, Monkey, Bovine, Dog

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser552 of mouse RelB protein.

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-RelB (Ser552) (D41B9) XP® Rabbit mAb #5025.


Transcription factors of the nuclear factor κB (NF-κB)/Rel family play a pivotal role in inflammatory and immune responses (1,2). There are five family members in mammals: RelA, c-Rel, RelB, NF-κB1 (p105/p50), and NF-κB2 (p100/p52). Both p105 and p100 are proteolytically processed by the proteasome to produce p50 and p52, respectively. Rel proteins bind p50 and p52 to form dimeric complexes that bind DNA and regulate transcription. In unstimulated cells, NF-κB is sequestered in the cytoplasm by IκB inhibitory proteins (3-5). NF-κB-activating agents can induce the phosphorylation of IκB proteins, targeting them for rapid degradation through the ubiquitin-proteasome pathway and releasing NF-κB to enter the nucleus where it regulates gene expression (6-8). NIK and IKKα (IKK1) regulate the phosphorylation and processing of NF-κB2 (p100) to produce p52, which translocates to the nucleus (9-11).


1.  Baeuerle, P.A. and Henkel, T. (1994) Annu Rev Immunol 12, 141-79.

2.  Baeuerle, P.A. and Baltimore, D. (1996) Cell 87, 13-20.

3.  Haskill, S. et al. (1991) Cell 65, 1281-9.

4.  Thompson, J.E. et al. (1995) Cell 80, 573-82.

5.  Whiteside, S.T. et al. (1997) EMBO J 16, 1413-26.

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

7.  Scherer, D.C. et al. (1995) Proc Natl Acad Sci USA 92, 11259-63.

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

9.  Senftleben, U. et al. (2001) Science 293, 1495-9.

10.  Coope, H.J. et al. (2002) EMBO J 21, 5375-85.

11.  Xiao, G. et al. (2001) Mol Cell 7, 401-9.


Entrez-Gene Id 5971
Swiss-Prot Acc. Q01201


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.

13567
Phospho-RelB (Ser552) (D41B9) XP® Rabbit mAb (PE Conjugate)