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Purified Mouse Anti-PI3-Kinase
Purified Mouse Anti-PI3-Kinase
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.
Purified Mouse Anti-PI3-Kinase
Immunofluorescent staining of A549 (ATCC CCL-185) cells.  Cells were seeded in a 96 well imaging plate (Cat. No. 353219) 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 (Cat. No. 554001).  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 (see Recommended Assay Procedure).
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 (Cat. No. 353219) 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 (Cat. No. 554001).  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 (see Recommended Assay Procedure).
Product Details
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BD Transduction Laboratories™
Human (QC Testing), Mouse, Rat, Dog, Chicken (Tested in Development)
Mouse IgG2a
Human PI3-Kinase α subunit aa. 562-724
Western blot (Routinely Tested), Bioimaging, Immunohistochemistry, Immunoprecipitation (Tested During Development)
85 kDa
250 µg/ml
AB_397460
Aqueous buffered solution containing BSA, glycerol, and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at -20°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.

Recommended Assay Procedures

Bioimaging

1. Seed the cells in appropriate culture medium at ~10,000 cells per well in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219) and culture overnight.

2. Remove the culture medium from the wells, and fix the cells by adding 100 μl of BD Cytofix™ Fixation Buffer (Cat. No. 554655) to each well.  Incubate for 10 minutes at room temperature (RT).

3. Remove the fixative from the wells, and permeabilize the cells using either BD Perm Buffer III, 90% methanol, or Triton™ X-100:

a. Add 100 μl of -20°C 90% methanol or Perm Buffer III (Cat. No. 558050) to each well and incubate for 5 minutes at RT.

     OR

b. Add 100 μl 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 μl of 1× PBS.

5. Remove the PBS, and block the cells by adding 100 μl of BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) to each well. Incubate for 30 minutes at RT.

6. Remove the blocking buffer and add 50 μl 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 μl of 1× PBS.

8. Remove the PBS, and add the second step reagent at its optimally titrated concentration in 50 μl 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 μl of 1× PBS.

10. Remove the PBS, and counter-stain the nuclei by adding 200 μl per well of 2 μg/ml Hoechst 33342 (e.g., Sigma-Aldrich Cat. No. B2261) in 1× PBS to each well at least 15 minutes before imaging.

11. View and analyze the cells on an appropriate imaging instrument.

Bioimaging:  For more detailed information please refer to http://www.bdbiosciences.com/support/resources/protocols/ceritifed_reagents.jsp

Western blot:  For more detailed information please refer to http://www.bdbiosciences.com/pharmingen/protocols/Western_Blotting.shtml

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. Please refer to www.bdbiosciences.com/us/s/resources 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.
610046 Rev. 3
Antibody Details
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4/PI3-Kinase

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) p85α, 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.

610046 Rev. 3
Format Details
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Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
610046 Rev.3
Citations & References
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View product citations for antibody "610046" on CiteAb

Development References (5)

  1. Cantley LC, Auger KR, Carpenter C, et al. Oncogenes and signal transduction. Cell. 1991; 64(2):281-302. (Biology). View Reference
  2. Efendiev R, Yudowski GA, Zwiller J, et al. Relevance of dopamine signals anchoring dynamin-2 to the plasma membrane during Na+,K+-ATPase endocytosis. J Biol Chem. 2002; 277(46):44108-44114. (Clone-specific: Immunofluorescence, Immunoprecipitation, Western blot). View Reference
  3. Kihara T, Shimohama S, Sawada H, et al. alpha 7 nicotinic receptor transduces signals to phosphatidylinositol 3-kinase to block A beta-amyloid-induced neurotoxicity. J Biol Chem. 2001; 276(17):13541-13546. (Clone-specific: Immunoprecipitation). View Reference
  4. Nguyen MH, Ho JM, Beattie BK, Barber DL. TEL-JAK2 mediates constitutive activation of the phosphatidylinositol 3'-kinase/protein kinase B signaling pathway. J Biol Chem. 2001; 276(35):32704-32713. (Clone-specific: Immunoprecipitation, Western blot). View Reference
  5. Zhang XA, Bontrager AL, Hemler ME.. Transmembrane-4 superfamily proteins associate with activated protein kinase C (PKC) and link PKC to specific beta(1) integrins. J Biol Chem. 2001; 276(27):25005-25013. (Clone-specific: Immunofluorescence, Immunoprecipitation). View Reference
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610046 Rev. 3

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For Research Use Only. Not for use in diagnostic or therapeutic procedures.