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14228S 100 µl (50 tests) $299.00.0
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REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R Endogenous Rabbit IgG
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Flow Cytometry

Flow cytometric analysis of serum-starved SK-N-MC cells, untreated (blue) or IBMX and Forskolin #3828-treated (0.5 mM and 30 μM, 30 min; green), using Phospho-CREB (Ser133) (87G3) 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-CREB (Ser133) (87G3) Rabbit mAb (PE Conjugate) detects endogenous levels of CREB only when phosphorylated at Ser133. The antibody also detects the phosphorylated form of the CREB-related protein, ATF-1.


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

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser133 of human CREB 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-CREB (Ser133) (87G3) Rabbit mAb #9198.


CREB is a bZIP transcription factor that activates target genes through cAMP response elements. CREB is able to mediate signals from numerous physiological stimuli, resulting in regulation of a broad array of cellular responses. While CREB is expressed in numerous tissues, it plays a large regulatory role in the nervous system. CREB is believed to play a key role in promoting neuronal survival, precursor proliferation, neurite outgrowth, and neuronal differentiation in certain neuronal populations (1-3). Additionally, CREB signaling is involved in learning and memory in several organisms (4-6). CREB is able to selectively activate numerous downstream genes through interactions with different dimerization partners. CREB is activated by phosphorylation at Ser133 by various signaling pathways including Erk, Ca2+, and stress signaling. Some of the kinases involved in phosphorylating CREB at Ser133 are p90RSK, MSK, CaMKIV, and MAPKAPK-2 (7-9).


1.  Lonze, B.E. et al. (2002) Neuron 34, 371-85.

2.  Lee, M.M. et al. (1999) J Neurosci Res 55, 702-12.

3.  Redmond, L. et al. (2002) Neuron 34, 999-1010.

4.  Dash, P.K. et al. (1990) Nature 345, 718-21.

5.  Yin, J.C. et al. (1994) Cell 79, 49-58.

6.  Guzowski, J.F. and McGaugh, J.L. (1997) Proc Natl Acad Sci USA 94, 2693-8.

7.  Xing, J. et al. (1998) Mol Cell Biol 18, 1946-55.

8.  Ribar, T.J. et al. (2000) J Neurosci 20, RC107.

9.  Tan, Y. et al. (1996) EMBO J 15, 4629-42.


Entrez-Gene Id 1385
Swiss-Prot Acc. P16220


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.

14228
Phospho-CREB (Ser133) (87G3) Rabbit mAb (PE Conjugate)