Flow cytometry. Immunoprecipitation. Immunohistochemistry. (1,2,3) Other applications not tested. Optimal dilutions are dependent on conditions and should be determined by the user.
Protocol
FLOW CYTOMETRY ANALYSIS: Method: 1. Prepare a cell suspension in media A. For cell preparations, deplete the red blood cellpopulation with Lympholyte®-M cell separation medium. 2. Wash 2 times. 3. Resuspend the cells to a concentration of 2x10e7 cells/ml in media A. Add 50 µl of thissuspension to each tube (each tube will then contain 1 x 10e6 cells, representing 1 test). 4. To each tube, add ~1. 0 µg* of this Ab per 10e6 cells. 5. Vortex the tubes to ensure thorough mixing of antibody and cells. 6. Incubate the tubes for 30 minutes at 4°C. 7. Wash 2 times at 4°C. 8. Add 100 µl of secondary antibody (Streptavidin-FITC) at a 1: 500 dilution. 9. Incubate tubes at 4°C for 30 - 60 minutes (It is recommended that tubes are protectedfrom light since most fluorochromes are light sensitive). 10. Wash 2 times at 4°C. 11. Resuspend the cell pellet in 50 µl ice cold media B. 12. Transfer to suitable tubes for flow cytometric analysis containing 15 µl of propidiumiodide at 0. 5 mg/ml in PBS. This stains dead cells by intercalating in DNA. Media: A. Phosphate buffered saline (pH 7. 2) + 5% normal serum of host species + sodium azide(100 µl of 2M sodium azide in 100 mls). B. Phosphate buffered saline (pH 7. 2) + 0. 5% Bovine serum albumin + sodium azide (100µl of 2M sodium azide in 100 mls). Results - Tissue Distribution: Mouse Strain: BALB/cCell Concentration: 1x10e6 cells per testsAntibody Concentration Used: 1. 0 µg/10e6 cellsIsotypic Control: Biotin Rat IgG2bCell Source: Percentage of cells stained above control: TK1 Cells: 97. 9%Thymus: 80. 0%Spleen: 85. 1%Bone Marrow: 72. 4%
Restrictions
For Research Use only
Concentration
0.1 mg/mL
Buffer
PBS, 0.02 % NaN3 and EIA grade BSA as a stabilizing protein to bring total protein concentration to 4-5 mg/mL
Preservative
Sodium azide
Precaution of Use
This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Handling Advice
Avoid repeated freezing and thawing.
Storage
4 °C/-20 °C
Storage Comment
Store the antibody undiluted at 2-8 °C for one month or (in aliquots) at -20 °C for longer.
Integrin alpha 4 (also called CD49d) is a 150 kDa protein that possesses a large extracellular domain involved in ligand binding, a single transmembrane domain, and an intracellular regulatory domain possessing multiple sites for phosphorylation. Integrin alpha 4 forms heterodimers with integrins beta 1 and beta 7. Integrin alpha 4 is expressed on leukocytes and leukocyte precursors, neural crest cells, and developing skeletal muscles and is essential for embryogenesis, hematopoiesis, and immune responses. The presence of integrin alpha 4 promotes cell migration and inhibits cell spreading and contractility. Integrin alpha 4 function has been implicated in the pathogenesis of multiple diseases including asthma, rheumatoid arthritis, Crohn's disease, ulcerative colitis, hepatitis C, and multiple sclerosis, and therefore, modulation of integrin alpha 4 function has become an important target for drug discovery.Synonyms: CD49 antigen-like family member D, Integrin alpha-4, Integrin alpha-IV, VLA-4, VLA4