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REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous 150: alpha9, 130: beta1 Mouse IgG1
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Immunoprecipitation

Immunoprecipitation of α9/β1 integrin from CHO cells transfected with human α9 integrin. Endogenous β1 integrin was detected by western blot using Integrin β1 Antibody #4706.

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Flow Cytometry

Flow cytometric analysis of CHO cells, untransfected (blue) or transfected with human α9 integrin (green), using Integrin α9β1 (Y9A2) Mouse mAb compared to a nonspecific negative control antibody (red).

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Immunoprecipitation for Native Proteins

This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity.

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 of 1X PBS, add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.

    NOTE: Add 1 mM PMSF (#8553) immediately prior to use.

  3. 3X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer.
  4. Protein G Magnetic Beads: Use Protein G (#8740) for mouse IgG immunoprecipitation.
  5. 6-Tube Magnetic Separation Rack: (#7017).
  6. 10X Kinase Buffer (for kinase assays): (#9802) To Prepare 1 ml of 1X kinase buffer, add 100 µl 10X kinase buffer to 900 µl dH2O, mix.
  7. ATP (10 mM) (for kinase assays): (#9804) To prepare 0.5 ml of ATP (200 µM), add 10 µl ATP (10 mM) to 490 µl 1X kinase buffer.

B. Preparing Cell Lysates

  1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
  2. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS.
  3. Remove PBS and add 0.5 ml ice-cold 1X cell lysis buffer to each plate (10 cm) and incubate on ice for 5 min.
  4. Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice.
  5. Sonicate on ice three times for 5 sec each.
  6. Microcentrifuge for 10 min at 4°C, 14,000 x g and transfer the supernatant to a new tube. The supernatant is the cell lysate. If necessary, lysate can be stored at -80°C.

C. Immunoprecipitation

Cell Lysate Pre-Clearing (Optional)

  1. Vortex to mix beads
  2. Add 10-30 µl of 50% Protein G magnetic bead slurry to 200 µl cell lysate at 1 mg/ml.
  3. Incubate with rotation at 4°C for 30-60 min.
  4. Pellet beads using magnetic separation rack. Transfer the supernatant to a fresh tube.
  5. Proceed to immunoprecipitation below.

Immunoprecipitation

  1. Add primary antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µl cell lysate at 1 mg/ml. Incubate with rotation overnight at 4°C.
  2. Add protein G magnetic beads (10-30 µl of 50% bead slurry). Incubate with rotation for 10-30 min at 4°C.
  3. Pellet beads using magnetic separation rack. Wash pellet five times with 500 µl of 1X cell lysis buffer. Keep on ice between washes.
  4. Proceed to analyze by western immunoblotting or kinase activity (section D).

D. Sample Analysis

Proceed to one of the following specific set of steps.

For Analysis by Western Immunoblotting

  1. Resuspend the pellet with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec at 14,000 x g.
  2. Heat the sample to 95-100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
  3. Load the sample (15-30 µl) on a 4-20% gel for SDS-PAGE.
  4. Analyze sample by western blot (see Western Immunoblotting Protocol).

For Analysis by Kinase Assay

  1. Wash pellet twice with 500 µl 1X kinase buffer. Keep on ice.
  2. Suspend pellet in 40 µl 1X kinase buffer supplemented with 200 µM ATP and appropriate substrate.
  3. Incubate for 30 min at 30°C.
  4. Terminate reaction with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec.
  5. Transfer supernatant containing phosphorylated substrate to another tube.
  6. Heat the sample to 95-100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
  7. Load the sample (15-30 µl) on SDS-PAGE (4-20%).

posted December 2008

revised November 2013

Protocol Id: 121
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Flow Cytometry, Methanol Permeabilization Protocol for Mouse Antibodies

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.
  5. Recommended Anti-Mouse secondary antibodies::

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

NOTE: Count cells using a hemocytometer or alternative method.

  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 primary antibody (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 100 µl of diluted fluorochrome-conjugated secondary antibody (prepared in incubation buffer at recommended dilution).
  7. Incubate for 30 min at room temperature.
  8. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  9. 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 June 2005

revised June 2017

Protocol Id: 406

Product Usage Information

Application Dilutions
Immunoprecipitation 1:50
Flow Cytometry 1:400

Storage: Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

Integrin α9β1 (Y9A2) Mouse mAb detects endogenous levels of total α9/β1 integrin heterodimer.


Species Reactivity: Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with murine L cells transfected with human α9 integrin protein.

Integrins are transmembrane glycoproteins that form heterodimers consisting of one α and one β subunit. The dimers act as receptors for extracellular matrix (ECM) proteins at sites of cell adhesion, and interact with focal adhesion (FA) proteins on the cytosolic side, forming the connection between the ECM and the actin cytoskeleton. Signaling to and from integrins regulates cell adhesion, motility, proliferation, apoptosis and gene expression, impacting cellular processes such as development, wound healing, immune response, invasion, metastasis and angiogenesis (reviewed in 1,2). α9β1 integrin is expressed in epithelial cells, smooth and skeletal muscle, neutrophils and hepatocytes (3). Its ligands include the ECM protein tenascin (4) and vascular cell adhesion molecule-1 (VCAM-1) (5). The cytoplasmic domain of α9 integrin binds the focal adhesion adaptor protein, paxillin, inhibiting cell spreading (6,7). Binding of the α9 cytoplasmic domain to spermidine/spermine N(1)-acetyltransferase (SSAT) mediates α9/β1 enhancement of cell migration (8). Physiological functions include development of the lymphatic system (9), possibly through binding to the lymphatic vascular endothelial growth factors VEGF-C and -D (10), neutrophil migration (5), and myogenic differentiation (11).


1.  Calderwood, D.A. et al. (2000) J Biol Chem 275, 22607-10.

2.  ffrench-Constant, C. and Colognato, H. (2004) Trends Cell Biol 14, 678-86.

3.  Palmer, E.L. et al. (1993) J. Cell Biol. 123, 1289-1297.

4.  Yokosaki, Y. et al. (1994) J. Biol. Chem. 269, 26691-26696.

5.  Taooka, Y. et al. (1999) J. Cell Biol. 145, 413-420.

6.  Young, B.A. et al. (2001) Mol. Biol. Cell 12, 3214-3225.

7.  Liu, S. et al. (2001) J. Biol. Chem. 276, 37086-37092.

8.  Chen, C. et al. (2004) J. Cell Biol. 167, 161-170.

9.  Huang, X.Z. et al. (2000) Mol. Cell Biol. 20, 5208-5215.

10.  Vlahakis, N.E. et al. (2005) J. Biol. Chem. 280, 4544-4552.

11.  Lafuste, P. et al. (2005) Mol. Biol. Cell 16, 861-870.


Entrez-Gene Id 3688 , 3680
Swiss-Prot Acc. P05556 , Q13797


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

4703
Integrin α9β1 (Y9A2) Mouse mAb