Upstream / Downstream

pathwayImage

Explore pathways related to this product.

To Purchase # 72423S

72423S 100 µl (50 tests) $299.00.0
$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 HeLa cells using IDH1 (D2H1) Rabbit mAb (PE Conjugate) (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed line).

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 antibody. Protect from light. Do not freeze.

Specificity / Sensitivity

IDH1 (D2H1) Rabbit mAb (PE Conjugate) recognizes endogenous levels of total IDH1 protein. This antibody does not recognize endogenous IDH2 protein, but does recognize recombinant levels of IDH2.


Species Reactivity: Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg222 of human IDH1 protein.

Product Description

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometric analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated IDH1 (D2H1) Rabbit mAb #8137.


IDH1 is one of three isocitrate dehydrogenases that catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG). These enzymes exist in two distinct subclasses that utilize either NAD or NADP+ respectively, as an electron acceptor (1). IDH1 is the NADP+-dependent isocitrate dehydrogenase found in the cytoplasm and peroxisomes. IDH2 and 3 are mitochodrial enzymes that also function in the Krebs cycle. IDH1 is inactivated by phosphorylation at Ser113 and contains a clasp-like domain wherein both polypeptide chains in the dimer interlock (2,3). IDH1 is expressed in a wide range of species and also in organisms that lack a complete citric acid cycle. Recently, an inactivating mutation of IDH1 has been implicated in glioblastoma (4). IDH1 appears to function as a tumor suppressor that, when mutationally inactivated, contributes to tumorigenesis in part through induction of the HIF-1 pathway (5).


1.  Ramachandran, N. and Colman, R.F. (1980) J Biol Chem 255, 8859-64.

2.  Bennett, P.M. and Holms, W.H. (1975) J Gen Microbiol 87, 37-51.

3.  Hurley, J.H. et al. (1990) Science 249, 1012-6.

4.  Bleeker, F.E. et al. (2009) Hum Mutat 30, 7-11.

5.  Zhao, S. et al. (2009) Science 324, 261-5.


Entrez-Gene Id 3417
Swiss-Prot Acc. O75874


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.

72423
IDH1 (D2H1) Rabbit mAb (PE Conjugate)