PI3-Kinase (AA 562-724) antibody

Antigen: PI3-Kinase

Binding Site: AA 562-724

Clonality: Monoclonal (4)

Host: Mouse

Reactivity: Human

Application: Western Blotting (WB), Immunohistochemistry (IHC), Immunoprecipitation (IP), BioImaging (BI)

Catalog no.: ABIN967707

Additional Information

Immunogen: Human PI3-Kinase alpha subunit

Cross-Reactivity: Dog (Canine), Rat (Rattus), Mouse (Murine), Chicken

Format: Liquid

Isotype: IgG2a, kappa

Clone: 4

Description: PI3-kinase phosphorylates the D-3 position of the inositol ring of phosphatidylinositol (PtdIns), PtdIns(4)P and PtdIns(4,5)P2 to produce the respective PI3-phosphorylated derivatives. PI3-kinase exists as a heterodimer of 85 kDa (p85) and 110 kDa (p110) subunits. The p85 subunit contains two SH2 domains and an SH3 domain. It associates with and serves as a substrate for activated growth factor receptor tyrosine kinases. p85 may serve as regulator of the catalytic subunit, p110, by acting as the link between PI3-kinase and the ligand-activated receptor. Two distinct forms of the p85 subunit have been described: 1) p85alpha, which binds tightly to the catalytic subunit, and 2) p85ß, a protein whose function is presently unknown. Both isoforms bind to activated receptors and serve as tyrosine kinase substrates.

Characteristics: 1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2. Please refer to us for technical protocols.
3. This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
4. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
5. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
6. Triton is a trademark of the Dow Chemical Company.

Molecular Weight: 85 kDa

Comments:

Related Products: ABIN967389, ABIN968533

Application Details

Application Notes: Bioimaging
1. Seed the cells in appropriate culture medium at ~10,000 cells per well in an 96-well Imaging Plate and culture overnight.
2. Remove the culture medium from the wells, and fix the cells by adding 100 myl of Fixation Buffer to each well. Incubate for 10 minutes at room temperature (RT).
3. Remove the fixative from the wells, and permeabilize the cells using either 90% methanol, or Triton™ X-100: a. Add 100 myl of -20°C 90% methanol to each well and incubate for 5 minutes at RT. OR b. Add 100 myl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.
4. Remove the permeabilization buffer, and wash the wells twice with 100 myl of 1× PBS.
5. Remove the PBS, and block the cells by adding 100 myl of to each well. Incubate for 30 minutes at RT.
6. Remove the blocking buffer and add 50 myl of the optimally titrated primary antibody (diluted in Stain Buffer) to each well, and incubate for 1 hour at RT.
7. Remove the primary antibody, and wash the wells three times with 100 myl of 1× PBS.
8. Remove the PBS, and add the second step reagent at its optimally titrated concentration in 50 myl to each well, and incubate in the dark for 1 hour at RT.
9. Remove the second step reagent, and wash the wells three times with 100 myl of 1× PBS.
10. Remove the PBS, and counter-stain the nuclei by adding 200 myl per well of 2 myg/ml Hoechst 33342 in 1× PBS to each well at least 15 minutes before imaging.
11. View and analyze the cells on an appropriate imaging instrument.

Concentration: 250 µg/ml

Purity: Purified

Purification: Purified from tissue culture supernatant or ascites by affinity chromatography.

Buffer: Aqueous buffered solution containing BSA, glycerol.

Preservative: 0.09% Sodium azide.

Storage: Store undiluted at -20° C.

Restrictions: For Research Use only

Images

Western blot analysis of PI3-Kinase on a A431 lysate. Lane 1: 1:2500, lane 2: 1:5000, lane 3: 1:10000 dilution of the PI3-Kinase antibody.

Immunofluorescent staining of A549 (ATCC CCL-185) cells. Cells were seeded in a 96 well imaging plate at ~ 10 000 cells per well. After overnight incubation, cells were stained using the alcohol perm protocol and the anti-PI3-Kinase antibody. The second step reagent was FITC goat anti mouse Ig. The image was taken on a BD Pathway™ 855 Bioimager using a 20x objective. This antibody also stained U-2 OS (ATCC HTB-96) and HeLa (ATCC CCL-2) cells using both the Triton™ X-100 and alcohol perm protocols.

Publications

Product: Cantley, Auger, Carpenter et al.: "Oncogenes and signal transduction." in: Cell, Vol. 64, Issue 2, pp. 281-302, 1991 (PubMed).

Kihara, Shimohama, Sawada et al.: "alpha 7 nicotinic receptor transduces signals to phosphatidylinositol 3-kinase to block A beta-amyloid-induced neurotoxicity." in: The Journal of biological chemistry, Vol. 276, Issue 17, pp. 13541-6, 2001 (PubMed).

Zhang, Bontrager, Hemler: "Transmembrane-4 superfamily proteins associate with activated protein kinase C (PKC) and link PKC to specific beta(1) integrins." in: The Journal of biological chemistry, Vol. 276, Issue 27, pp. 25005-13, 2001 (PubMed).

Nguyen, Ho, Beattie et al.: "TEL-JAK2 mediates constitutive activation of the phosphatidylinositol 3'-kinase/protein kinase B signaling pathway." in: The Journal of biological chemistry, Vol. 276, Issue 35, pp. 32704-13, 2001 (PubMed).

Efendiev, Yudowski, Zwiller et al.: "Relevance of dopamine signals anchoring dynamin-2 to the plasma membrane during Na+,K+-ATPase endocytosis." in: The Journal of biological chemistry, Vol. 277, Issue 46, pp. 44108-14, 2002 (PubMed).