This method facilitates the effective characterization of disease related RAF kinase methods. This technique is highly sensitive and reproducible, cost effective, user friendly, and does not require any advanced equipment. To measure the catalytic activity of RAF mutants, introduce flag tech mutations into the RAF coding sequence by PCR, before inserting whole sequences into the appropriate vector according to standard protocols.
Insert the glutathione as transferase coding sequence upstream of the RAF mutant coding sequences, to generate vectors encoding glutathione as transferase fused RAF mutants, in the same manner. Transvect 80%to 90%confluent 293T cell cultures with the vectors encoding the flag tagged RAF kinases or their mutants, according to standards protocols. After 24 hours of culture, replace the medium in each culture.
After 48 hours of culture, add 400 microliters of lysis buffer supplemented with protease and phosphatase inhibitors, to each well on ice. After five to 10 minutes transfer the cell lysates to a 1.5 milliliter tube for centrifugation to sediment the cell debris. Transfer 300 microliters of each sample into individual 1.5 milliliter micro centrifuge tubes.
Set aside 40 microliters per sample for downstream wholesale lysate fossil extra cellular signal regulated kinase one and two expression and activity amino blot analysis. Add 20 microliters of anti-FLAG affinity beads to each tube, for a one hour incubation with rotation in a four degrees Celsius cold room. At the end of the incubation quickly and gently wash the anti flag beads one time with lysis buffer in the cold room and one time with kinase reaction buffer before adding 20 microliters of kinase reaction mixture.
Graph Mutants with a low Dima affinity may lose their catalytic activity in vitro because of Dima dissociation. Therefore, the quick and gentle wash is critical. After 10 minutes at room temperature with flipping every other minute, stop the kinase reactions with five microliters of five XSDS sample buffer, per sample and boil the samples at 90 degrees Celsius for five minutes.
At the end of the incubation, run the samples on a nine to 12%polyacrylamide electrophoresis gel with 0.1%SDS, then transfer the proteins to a nitrocellulose membrane to allow detection of the levels of phospho mitogen-activated protein kinase and RAF mutants in each sample, by immuno blotting. To evaluate the Allosteric activity of kinase dead RAF mutants, construct vectors encoding the RAF receiver or the kinase dead RAF activators. Transvect 293 T cells with two vectors encoding both the RAF receiver and the kinase dead RAF activator, or a single vector encoding one of the proteins as demonstrated.
Harvest the whole cell lysates after 48 hours of culture as demonstrated, and quickly mix the clean whole cell lysate with five XSDS sample buffer at room temperature, Before boiling the lysates for five minutes at 90 degrees Celsius. Then run the boiled whole cell lysate samples on a nine to 12%polyacrylamide electrophoresis gel that's 0.1%SDS and transfer the proteins to a nitrocellulose membrane for detecting the levels of phospho extra cellular signal regulated kinase one and two and control proteins by immune blotting as demonstrated. To measure the relative dimer affinity and or stability of the RAF mutants construct vectors encoding flag tech draft mutants fused to the terminus of Firefly luciferase or the C Terminus a firefly luciferase.
Transvect 293 T cells with a pair of vectors encoding different and Terminus of firefly luciferase RAF mutants and C Terminus a firefly luciferase RAF mutants as demonstrated. 24 hours after the transvection replate two times 10 to the fifth 293 T cell transactions per well into crystal black image plates in color free medium. 48 hours after the transvestion add the luciferin to 293 T cell transvectents and incubate for 30 minutes.
Before measuring the luciferase signals on a multi detection system. At the end of the measurement aspirate the supernatents, and blice the transacted 293 T cells with lysas buffer as demonstrated. Run the whole cell lysate samples on a nine to 12%polyacrylamide electrophoresis gel with 0.1%SDS.
Detect the expression levels of the end terminus of Firefly luciferase RAF mutants, or the C terminus of Firefly luciferase RAF mutants by anti flag amino blot as demonstrated. Then normalize the luciferase signals of the 293 T cell transvectants according to the expression levels of the end terminus of Firefly luciferase and C terminus of Firefly luciferase RAF mutants. The in vitro kinase asa can be used to effectively probe the catalytic activity of some constitutively active R-spine mutants, but not that of kinase dead mutants with a fused C-spine.
The catalytic activity of constitutively active RAF mutants with a weak dimer affinity or stability however, might not be probed by using this ASA as their dimers are broken during the purification process. To avoid the loss of catalytic activity of RAF mutants with weak dimer affinity or stability, these mutants can be fused with glutothionas transferase to rescue the catalytic activity of the mutants. The RAF co-activation ASA can be used to evaluate the allosteric activity of kinase dead RAF Mutants.
As illustrated kinase dead mutants with an acidic end terminal motif and C spine fusion, function as allosteric activators to trigger the catalytic activity of mutants with a non phosphor relatable acidic end terminal motif, when co expressed in 293T cells. Further a complimentary split luciferase SA can be used for measuring the relative dimer affinity or stability of RAF family kinases and their mutants. Using a lysas buffer with a low concentration of detergent, and a quick gentle wash procedure during the sample purification is essential for a successful in vitro kinase SA.This technique will help researchers determine precisely how disease relay the RAF mutants, activate the downstream pathway, facilitating the selection of appropriate therapeutic strategies for disease treatment.
