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8864
IGF-I Receptor β (D23H3) XP® Rabbit mAb (PE Conjugate)
Antibody Conjugates

IGF-I Receptor β (D23H3) XP® Rabbit mAb (PE Conjugate) #8864

APPLICATIONS

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

Flow cytometric analysis of SK-UT-1 cells (blue) and MCF-7 cells (green) using IGF-I Receptor β (D23H3) XP® Rabbit mAb (PE Conjugate) (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed lines).

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Flow Cytometry Triton™ X-100 Permeabilization Protocol - Conjugated Antibody

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. Adjust pH to 8.0.
  2. Formaldehyde (methanol free).
  3. Incubation Buffer (1X PBS / 0.5% BSA): Dissolve 0.5 g bovine serum albumin (BSA) in 100 mL 1X PBS. Store at 4°C.
  4. Permeabilization Buffer (1X PBS / 0.3% Triton™ X-100 / 0.5% BSA): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml Incubation Buffer.

B. Fixation and Permabilization

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 briefly in 0.5-1 ml PBS. Add formaldehyde to a final concentration of 2-4% formaldehyde.
  3. Fix for 15 minutes at room temperature (20-25 °C).
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container.
  5. Resuspend cells in 1 ml Permeabilization Buffer.
  6. Incubate for 10 minutes at room temperature.
  7. Proceed with staining or store cells at +4°C in PBS with 0.1% sodium azide.

C. Staining Using Conjugated Primary Antibody

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells (suggested range of 0.5x106 to 1x106 cells).
  2. Centrifuge cells and discard supernatant.
  3. Resuspend cells in 100 µl of diluted conjugated primary antibody (prepared in Incubation Buffer at the recommended dilution).
  4. Incubate for 30-60 min at room temperature (20-25 °C). Protect from light.
  5. Wash by centrifugation in 1X PBS. Discard supernatant. Repeat.
  6. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section D).

D. Optional DNA Dye

  1. Resuspend cells in 0.1-0.5 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 January 2017

revised May 2018

Protocol Id: 1344

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.

IGF-I Receptor β (D23H3) XP® Rabbit mAb (PE Conjugate) recognizes endogenous levels of total IGF-I receptor β protein. This antibody does not cross-react with insulin receptor.

Species Reactivity:

Human, Mouse, Rat, Monkey

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human IGF-I receptor β protein.

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 IGF-I Receptor β (D23H3) XP® Rabbit mAb #9750.

Type I insulin-like growth factor receptor (IGF-IR) is a transmembrane receptor tyrosine kinase that is widely expressed in many cell lines and cell types within fetal and postnatal tissues (1-3). Receptor autophosphorylation follows binding of the IGF-I and IGF-II ligands. Three tyrosine residues within the kinase domain (Tyr1131, Tyr1135, and Tyr1136) are the earliest major autophosphorylation sites (4). Phosphorylation of these three tyrosine residues is necessary for kinase activation (5,6). Insulin receptors (IRs) share significant structural and functional similarity with IGF-I receptors, including the presence of an equivalent tyrosine cluster (Tyr1146/1150/1151) within the kinase domain activation loop. Tyrosine autophosphorylation of IRs is one of the earliest cellular responses to insulin stimulation (7). Autophosphorylation begins with phosphorylation at Tyr1146 and either Tyr1150 or Tyr1151, while full kinase activation requires triple tyrosine phosphorylation (8).

  1. Adams, T.E. et al. (2000) Cell Mol Life Sci 57, 1050-93.
  2. Baserga, R. (2000) Oncogene 19, 5574-81.
  3. Scheidegger, K.J. et al. (2000) J Biol Chem 275, 38921-8.
  4. Hernández-Sánchez, C. et al. (1995) J Biol Chem 270, 29176-81.
  5. Lopaczynski, W. et al. (2000) Biochem Biophys Res Commun 279, 955-60.
  6. Baserga, R. (1999) Exp Cell Res 253, 1-6.
  7. White, M.F. et al. (1985) J Biol Chem 260, 9470-8.
  8. White, M.F. et al. (1988) J Biol Chem 263, 2969-80.
Entrez-Gene Id
3480
Swiss-Prot Acc.
P08069
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.

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To Purchase # 8864S

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Product # Size Price
8864S
100 µl  (50 tests) $ 348