Upstream / Downstream

pathwayImage

Explore pathways related to this product.

Antibody Guarantee

CST Antibody Performance Guarantee

LEARN MORE  

To Purchase # 72115S

72115S 100 µl (50 tests) $329.00.0
$0.00

Questions?

Find answers on our FAQs page.

ANSWERS  

PhosphoSitePlus® Resource

  • Additional protein information
  • Analytical tools

LEARN MORE

REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R Mk Endogenous Rabbit IgG
Image
Image
Image
Image

Flow Cytometry

Flow cytometric analysis of PC-12 cells using GSK-3β (D5C5Z) XP® Rabbit mAb (PE Conjugate) (green) compared to Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (red).

Learn more about how we get our images
Image
Image
Page

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

GSK-3β (D5C5Z) XP® Rabbit mAb (PE Conjugate) recognizes endogenous levels of total GSK-3β protein. This antibody does not cross-react with GSK-3α protein.


Species Reactivity: Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the carboxy terminus of human GSK-3β protein.

Product Description

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in rat cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated GSK-3β (D5C5Z) XP® Rabbit mAb #12456.


Glycogen synthase kinase-3 (GSK-3) was initially identified as an enzyme that regulates glycogen synthesis in response to insulin (1). GSK-3 is a ubiquitously expressed serine/threonine protein kinase that phosphorylates and inactivates glycogen synthase. GSK-3 is a critical downstream element of the PI3K/Akt cell survival pathway whose activity can be inhibited by Akt-mediated phosphorylation at Ser21 of GSK-3α and Ser9 of GSK-3β (2,3). GSK-3 has been implicated in the regulation of cell fate in Dictyostelium and is a component of the Wnt signaling pathway required for Drosophila, Xenopus, and mammalian development (4). GSK-3 has been shown to regulate cyclin D1 proteolysis and subcellular localization (5).


1.  Welsh, G.I. et al. (1996) Trends Cell Biol 6, 274-9.

2.  Srivastava, A.K. and Pandey, S.K. (1998) Mol Cell Biochem 182, 135-41.

3.  Cross, D.A. et al. (1995) Nature 378, 785-9.

4.  Nusse, R. (1997) Cell 89, 321-3.

5.  Diehl, J.A. et al. (1998) Genes Dev 12, 3499-511.


Entrez-Gene Id 2932
Swiss-Prot Acc. P49841


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.

72115
GSK-3β (D5C5Z) XP® Rabbit mAb (PE Conjugate)