Innate defenses to viral infections are triggered by pattern recognition receptors or prrs. Present in cells of the innate immune system during an infection. Cytoplasmic PRRS rig eye and PKR bind to viral signature RNAs change confirmation all liga eyes and activate antiviral signaling to monitor rig eye and PKR confirmational changes and all ligamentization in vitro human A 5 49 cells are infected with Rift Valley fever virus.
Clone 13 to stimulate PRR activation. Cell lysates of control and infected cells are then prepared to analyze for rig eye and PKR confirmational changes. A limited tryin digestion is performed.
If confirmational changes in protein structure have occurred, the protein is more resistant to digestion and bands will be seen with Western blotting Analysis to analyze for formation of allgas native page is performed followed by western blotting analysis. If all ligamentization has occurred higher, all liga rig eye and PKR complexes are seen. The main advantage of limited protease digestion and native page is that these techniques facilitate the direct measurement of rega and PKR activation.
These methods can help answer key questions in the field of innate immunity, such as what is the exact nature and origin of the RNA species relevant for rig eye and PKR activation. These methods can provide insight into rig eye and PKR agonists. They can also be applied to other cytoplasmic sensor proteins such as MDA five, demonstrating the procedure will be Mila Viva, a PhD student from my laboratory Before beginning this procedure.
Please note that Rift Valley Fever virus clone 13 referred to hereafter as CL 13 is an attenuated virus mutant, which in Germany must be handled under BSL two conditions to prewarm PBS serum free medium and cell culture medium containing 5%FCS. Place them in a water bath in a 1.5 milliliter micro centrifuge tube. Prepare 1.25 times 10 to the seventh PFU per milliliter of CL 13 in serum free medium to infect 2.5 times 10 to the sixth.
A 5 49 cells with a multiplicity of infection or MOI of five prepare roughly 10%more than needed to account for pipetting errors. Remove the A 5 49 cells from the incubator, aspirate the medium and add 10 milliliters of PBS swirl or tip the culture flask to wash the cells and then remove the PBS. Next, add one milliliter of the diluted CL 13 or for the uninfected control or mock infection.
Add one milliliter of serum free medium and incubate for one hour every 15 minutes. Carefully tip the flask to ensure equal distribution of the medium. After one hour of infection, remove the inocular.
Add five milliliters of prewarm cell culture medium with 5%FCS, and incubate for five hours. Prepare PBS with 0.5%tritton X 100 and place it at four degrees Celsius. Do not add Syrian protease inhibitors.
Next, wash the cells with cold PBS and add 10 milliliters of fresh PBS. Then using a cell scraper, detach the cells from the dish. Transfer the cell suspension to a 15 milliliter conical tube and centrifuge at 800 times G for five minutes at room temperature.
After the spin, remove the supernatant and resuspend the cell pellet in 30 microliters of PBS with 0.5%Triton X 100. Transfer the lysate into a fresh 1.5 milliliter tube and incubate for at least 10 minutes on ice. Centrifuge the lysate at 10, 000 times G for 10 minutes at four degrees Celsius.
After the centrifugation, transfer the clarified cell lysate to a fresh tube. Perform a Bradford assay to determine the protein concentration in the clarified cell lysate Store at minus 20 degrees Celsius or proceed immediately to try and digestion or native page to assay for confirmational changes of pattern recognition receptors first dilute L one tosto chloro, methyl ketone treated or TPCK trypsin in PBS to a final working concentration of two micrograms per milliliter in two new tubes for each condition, adjust the final protein concentration of the CL 13 and non-infected control protein lysates to 25 micrograms in a final volume of nine microliters with PBS, one set of lysates will be used as an input control and the other will be treated with TPCK trypsin to the untreated control tubes. Add one microliter of PBS to the remaining two tubes.
Add one microliter of two micrograms per microliter TPCK trypsin to bring the final concentration to 0.2 micrograms per microliter. Mix the reactions by pipetting. Incubate the lysates at 37 degrees Celsius for 25 minutes.
Stop the reaction by adding five x denaturing sample buffer and then boiling for five minutes at 95 degrees Celsius. It is important to not extend the trypsin incubation time. Note that precise timing of the trypsin digestion is critical for detection of resistant fragments without any background if resistant proteins are not detected or if too many are detected, adjusted duration of digestion.Accordingly.
After boiling store the samples at minus 20 degrees Celsius or load the samples on two sodium docal sulfate. Poly acrylamide gels consisting of a 5%stacking gel over a 12%resolving gel. Separate the proteins at 25 milliamps per gel until the bromo phenol blue runs out.
After running the gels, transfer the proteins from one gel to a poly vine fluoride membrane and analyzed by western blotting with antibodies directed against rig eye and PKR stain the other gel with kumasi brilliant blue G two 50. Then store the gel in 25%ethanol, 8%acetic acid, and 4%glycerol at four degrees Celsius or proceed to perform imaging and analysis. To analyze oligomeric states of pattern recognition receptors prepare 50 micrograms of cell lysate in a final volume of 10 microliters with PBS and add five x sample buffer to a final concentration of one x.
Immediately load the samples on a native poly acrylamide gel with a 5%stacking and an 8%resolving gel. Any delay will result in a loss of native complexes. Run the gel at 20 milliamps per gel at four degrees Celsius.
Stop the electrophoresis 45 minutes after the Bromo phenol blue band has left the gel after a total of approximately 1.5 to two hours per perform Western blotting using antibodies directed against rig eye and PKR as described in the accompanying document to assay for confirmational switching and oligo induced by recognition of a viral agonist by rig eye or PKR limited protease digestion and native page were performed as described in this video, trypsin digestion of mock infected cell lysates resulted in a rapid degradation of rig eye, whereas clone 13 infection led to the generation of a 30 kilodalton resistant rig eye fragment. PKR also shows partial resistance to tripsin digestion in infected samples, which coincides with its phosphorylation to monitor efficiency and specificity of tripsin digestion gels were stained with kumasi brilliant blue G two 50. Untreated samples show equal amounts of loaded proteins, subjecting mock, and C 13 cell lysates to tripsin digestion leads to a comparable decrease of global protein amounts.
This demonstrates that tripsin treatment has the same efficiency for mock and CL 13 infected samples in non-infected cells. Only monomers of R EYE and PKR were detected as an additional control. The transcription factor IRF three was included, which is present as a monomer, but known to dimerize upon activation via R Eye.
Clone 13 infection leads to a strong accumulation of rig eye oligomeric complexes in the form of a smear and of PKR and IRF three dimers or oligomers as a defined protein band. These results demonstrate that limited trips in digestion and native page are useful tools to monitor confirmational changes and oligo formation of rig eye and PKR upon infection. After watching this video, you should have a good understanding of how to monitor WIC eye and PKR confirmation switching and ization once mastered.
This technique can be done in two days following this procedure. As a methods like pro assays can be performed in order to answer additional questions like whether there's a stable interaction with the stimulating ligand after its development. This technique paved the way for researchers in the field of innate immunity to explore the activation status of pattern recognition receptors in viral stimulated cells.
