|Reactivity||Human Mouse Rat|
|Storage buffer||10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide.|
|Storage||Store at –20°C.|
1. Aspirate media from cultures and Wash the cells with 1X PBS. 2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice. 3. Sonicate for 10–15 sec to complete cell lysis and shear DNA. 4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice. 5. Centrifuge for 5 min (with Microcentrifuge). 6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins). NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer. 7. Electrotransfer to nitrocellulose/PVDF membrane.
Membrane Blocking and Antibody Incubations
1. (Optional) After transfer, wash the transferred membrane with TBS for 5 min at room temperature. 2. Incubate the membrane in the blocking buffer for 1 hr at room temperature. 3. Wash three times for 5 min each with TBST.
b. Antibodies Incubation
1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C. 2. Wash three times for 5 min each with TBST. 3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature. 4. Wash three times for 5 min each with TBST. 5. Proceed with detection.
Detection of Proteins
1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST. 2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). Mix well. 3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.
|Specificity||Cortactin Rabbit Recombinant mAb detects endogenous level of total Cortactin.|
|Background||Cortactin is an actin-nucleation-promoting factor that is shown to colocalize with cortical F-actin at the cell periphery. It acts downstream of E-cadherin activated by Src kinase and is required for actin polymerization at these cell-cell adhesions. Cortactin can activate the Arp2/3 complex and stabilize branched actin networks. In addition to interactions with its numerous binding partners, cortactin activity is also regulated by several post-translational modifications. Cortactin is phosphorylated at tyrosine residues in response to signaling downstream of different upstream receptors, which include integrin- and cadherin-adhesion receptors and growth factor receptors. Several nonreceptor tyrosine kinases have been implicated in the phosphorylation of cortactin, including Src family kinases, ABL family kinases, FER, and Syk. Cortactin is also phosphorylated by several serine/threonine kinases. It integrates signals from diverse upstream signaling cascades. In addition to phosphorylation, cortactin is also regulated through acetylation by histone acetyltransferase p300, and deacetylation by histone deacetylase 6 (HDAC6) and sirtuin-1 (SIRT1). Acetylation of cortactin regulates binding of F-actin. Cortactin is overexpressed in many cancers and is crucial for cancer cell invasion and metastasis. It is essential for the formation and function of invadopodia.|