|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.
Specimen Preparation (forcultured cell lines, IF-IC)
1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
1. Add theblocking buffer and incubate for 60 min at RT.
|Specificity||A-RAF Rabbit Recombinant mAb detects endogenous levels of total A-RAF.|
|Background||The Ras-Raf-MEK-MAPK (mitogen-activated protein kinase)-signaling pathway plays a key role in the regulation of many cellular functions, including cell proliferation, differentiation and transformation, by transmitting signals from membrane receptors to various cytoplasmic and nuclear targets. One of the key components of this pathway is the serine/threonine protein kinase, Raf. The Raf family kinases consist of three isoforms: A-Raf, B-Raf and C-Raf. B-Raf is highly expressed in neural tissue such as the brain and spinal cord, other tissues however express less B-Raf and its expression in muscle is barely detectable, whereas by contrast C-Raf is ubiquitously expressed with the exception of the brain. A-Raf has more limited tissue distribution, it is most highly expressed in the epididymis and ovaries and it is also present in abundance in liver, uterus and kidney. Notably, some of these are steroid hormone-secreting organs or play an important role in steroid hormone production, so it maybe that A-Raf could be involved in the regulation of steroid hormone synthesis and secretion or that steroid hormones and their receptors may regulate physical functions via A-Raf. A-Raf has a stable or transient localization to the mitochondria and tubular endosomes, which has implicated it in the regulation of apoptosis, energy balance and endocytic membrane traffic. In comparison with B-Raf and C-Raf, A-Raf has the lowest basal kinase activity, the maximal activity of A-RAF is only 20% of that of C-Raf. However, a number of publications have revealed that A-Raf plays an important role in apoptosis, tumorigenesis and resistance to Raf inhibitors. Regarding regulation of A-Raf isoform activity by phosphorylation. A-Raf is weakly activated by oncogenic Ras but more strongly by oncogenic Src and both of these two signals synergize to produce maximal activation. A-Raf was also shown to play a role in regulating the growth of human hematopoietic cells.|