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Western blot analysis of PI4-Kinase β on a HeLa lysate. Lane 1: 1:10000, lane 2: 1:20000, lane 3: 1:40000 dilution of the PI4-Kinase β 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-PI4 Kinase β 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 HeLa (ATCC CCL-2) and U-2 OS (ATCC HTB-96) cells using both the Triton™ X-100 and alcohol perm protocols (see Recommended Assay Procedure).
BD Transduction Laboratories™ Purified Mouse Anti-PI4-Kinase β
BD Transduction Laboratories™ Purified Mouse Anti-PI4-Kinase β
Regulatory Status Legend
Any use of products other than the permitted use without the express written authorization of Becton, Dickinson and Company is strictly prohibited.
Preparation And Storage
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
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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.
- 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.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Triton is a trademark of the Dow Chemical Company.
Companion Products
Phosphoinositide turnover is a well established mechanism of intracellular signal transduction. Sequential phosphorylation of phosphatidylinositol (PtdIns) results inPtdIns-4-phosphate (PIP) and PtdIns-4,5-bisphosphate (PIP2). Phospholipase C (PLC)hydrolyzes PIP2 to inositol-1,4,5-trisphosphate (IP3) which stimulates release of intracellular Ca2+. PIP is generated by phosphorylation of PtdIns at the D4 position of the inositol ring. This event is mediated by the PtdIns 4-kinases (PI4-K). These enzymes are divided into two types (II and III) based on their size and sensitivity to certain compounds. Although the PI4-Ks are abundantly distributed throughout the cell, activity is found primarily in association with membranous structures. Members of this family contain a lipid kinase unique domain and a C-terminal catalytic domain. Two mammalian PI4-Ks, PI4-Kα and PI4-Kß, have been identified. PI4-Kß is homologous to the yeast PI4-K, PIK1. Based on its size and sensitivity to wortmanin (a PI3-K inhibitor), PI4-Kß is classified as a type III enzyme. Although it is found in the cytosol and in association with the Golgi, the specific function of PI4-Kß is yet to be determined.
Development References (3)
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Balla T, Downing GJ, Jaffe H, Kim S, Zolyomi A, Catt KJ. Isolation and molecular cloning of wortmannin-sensitive bovine type III phosphatidylinositol 4-kinases. J Biol Chem. 1997; 272(29):18358-18366. (Biology). View Reference
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Meyers R, Cantley LC. Cloning and characterization of a wortmannin-sensitive human phosphatidylinositol 4-kinase. J Biol Chem. 1997; 272(7):4384-4390. (Biology). View Reference
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Wong K, Meyers ddR, Cantley LC. Subcellular locations of phosphatidylinositol 4-kinase isoforms. J Biol Chem. 1997; 272(20):13236-13241. (Biology). View Reference
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.