PTK2 Protein tyrosine Kinase 2 (PTK2) (AA 354-533) antibody

Antigen: PTK2 Protein tyrosine Kinase 2 (PTK2)

Binding Site: AA 354-533

Clonality: Monoclonal (77)

Host: Mouse

Reactivity: Chicken

Conjugate: Un-conjugated

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

Catalog no.: ABIN967736

Additional Information

Alternative name: FAK

Immunogen: Chicken FAK

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

Format: Liquid

Isotype: IgG1

Clone: 77

Description: Focal Adhesion Kinase (FAK) is a cytoplasmic tyrosine kinase that colocalizes with integrins in focal adhesions. This cellular localization is directed by a 125 amino acid sequence at the C-terminus called the Focal Adhesion Targeting sequence (FAT). The binding of extracellular matrix ligands to integrins triggers autophosphorylation and activation of FAK. This creates binding sites for SH2 domains of intracellular signaling molecules such as src, PI3 kinase, and Grb2. FAK's ability to bind numerous structural and signaling proteins via a variety of interactions has led to substantial speculation about its function. Although FAK's precise role has not been elucidated, proposed possibilities include regulating cell motility, cell growth, cytoskeletal organization, and adhesion-dependent cell survival.
Synonyms: Focal Adhesion Kinase

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: 116-125 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.

Research Area: Cancer, Extracellular Matrix, Tyrosine Kinases, Angiogenesis, Kinases/Phosphatases

Restrictions: For Research Use only

Images

Western blot analysis of FAK on a A431 cell lysate (Human epithelial carcinoma, ATCC CRL-1555). Lane 1: 2 µg/ml, lane 2: 1 µg/ml, lane 3: 0.5 µg/ml of the mouse anti- FAK antibody.

Immunofluorescent staining of U2OS (ATCC HTB-96) 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-FAK antibody. The second step reagent was FITC goat anti-mouse Ig. Images were taken on a BD Pathway™ 855 bioimaging system using a 20x objective. This antibody also stained A549 (ATCC CCL-185) and HeLa (ATCC CCL-2) cells and worked with both the Triton™ X-100 and alcohol perm protocols.

Publications

Product: Clancy, Rediske, Tang et al.: "Outside-in signaling in the chondrocyte. Nitric oxide disrupts fibronectin-induced assembly of a subplasmalemmal actin/rho A/focal adhesion kinase signaling complex." in: The Journal of clinical investigation, Vol. 100, Issue 7, pp. 1789-96, 1997 (PubMed).

Huan, van Adelsberg: "Polycystin-1, the PKD1 gene product, is in a complex containing E-cadherin and the catenins." in: The Journal of clinical investigation, Vol. 104, Issue 10, pp. 1459-68, 1999 (PubMed).

Kovacic-Milivojević, Roediger, Almeida et al.: "Focal adhesion kinase and p130Cas mediate both sarcomeric organization and activation of genes associated with cardiac myocyte hypertrophy." in: Molecular biology of the cell, Vol. 12, Issue 8, pp. 2290-307, 2001 (PubMed).

Kim, Feldman: "Insulin-like growth factor I prevents mannitol-induced degradation of focal adhesion kinase and Akt." in: The Journal of biological chemistry, Vol. 277, Issue 30, pp. 27393-400, 2002 (PubMed).

Zeng, Si, Yu et al.: "PTP alpha regulates integrin-stimulated FAK autophosphorylation and cytoskeletal rearrangement in cell spreading and migration." in: The Journal of cell biology, Vol. 160, Issue 1, pp. 137-46, 2003 (PubMed).

Jones, Stewart: "Nuclear import of N-terminal FAK by activation of the FcepsilonRI receptor in RBL-2H3 cells." in: Biochemical and biophysical research communications, Vol. 314, Issue 1, pp. 39-45, 2004 (PubMed).

Yi, Zhou, Huber et al.: "Nuclear compartmentalization of FAK and FRNK in cardiac myocytes." in: American journal of physiology. Heart and circulatory physiology, Vol. 290, Issue 6, pp. H2509-15, 2006 (PubMed).