|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.
Raf1 Antibody detects endogenous levels of total Raf1.
RAF family kinases primarily act as signalling relays downstream of RAS. The Raf-1 kinase (also known as c-Raf) lies at the heart of a signalling network that controls cell proliferation, neoplastic transformation, differentiation and apoptosis. Many of these effects are transmitted via the MAPK/ERK pathway, a three-tiered kinase cascade, where Raf-1 phosphorylates and activates MEK, which then phosphorylates and activates ERK. Activated Ras binds to Raf-1 with high affinity, but does not alter the catalytic activity of Raf-1 directly. Rather, it relocalizes Raf-1 from the cytosol to the plasma membrane where a multistep activation process takes place. Raf-1 activation entails interaction with modulatory proteins, lipids, and complex changes in phosphorylation. The phosphorylation of Ser339 and Tyr341 is absolutely required and co-operates in Raf-1 activation. Furthermore, phosphorylation of Thr491 and Ser494 in the activation loop is necessary, but not sufficient for activation. These sites co-operate with Ser338 and Tyr341. Raf-1 is also negatively regulated by phosphorylation. The cAMP-dependent kinase PKA inhibits Raf-1, phosphorylating Raf-1 on Ser43, Ser259 and Ser621. These sites are phosphorylated in resting cells, but are hyperinduced by PKA.