Immunofluorescence analysis of FAK was done on 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 15 minutes; permeabilized with 0.25% Triton™ X-100 for 10 minutes followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with FAK Mouse Monoclonal Antibody (AHO1272) at 1µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Flour 488 Rabbit Anti-Mouse IgG Secondary Antibody (A11059) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor 594 Phalloidin (A12381). Panel d is a merged image showing membrane and cytoplasmic FAK localization. Panel e shows no primary antibody control. The images were captured at 20X magnification.
|Tested species reactivity||Human, Mouse, Rat|
|Host / Isotype||Mouse / IgG2b, kappa|
|Immunogen||Recombinant fragment of human FAK expressed in E. coli.|
|Storage buffer||PBS, pH 7.2, with 1% BSA|
|Contains||0.1% sodium azide|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1-3 µg/10^6 cells|
|Immunocytochemistry (ICC)||1-2 ug/ml|
|Immunofluorescence (IF)||1-2 ug/ml|
|Immunohistochemistry (Paraffin) (IHC (P))||1:10-1:100|
|Western Blot (WB)||1-2 ug/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Focal Adhesion Kinase (FAK) is a 125 kDa non-receptor protein tyrosine kinase that acts as a substrate for Src and is a key element of integrin signaling. FAK plays an important role in cell spreading, differentiation, migration, cell death, and acceleration of the G1 to S phase transition of the cell cycle. Tyrosine 397 is the autophosphorylation site of FAK, and involved in its initial activation. This phosphorylated site binds Src family SH2 domains and the p85 subunit of PI3-Kinase, and activates cell migration and invasion.
IP-MS enrichment of FAK (LFQ intensity): FAK was enriched 290-fold from LNCAP lysate compared to background proteins, using the optimized IP-MS workflow with Pierce MS-Compatible Magnetic IP Kit protein A/G (Part No. 90409) and FAK antibody (Part No. AHO1272). See more information on IP-MS verification of antibody selectivity. IP-MS validation info.
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