This procedure begins by removing any debris from the influenza. A virus solution by centrifugation. The viral membrane is then disrupted with the detergent and the release to VR nps are separated on a glycerol gradient.
Centrifugation fractions from the gradient are collected and an aliquot of each fraction is run. On an SDS page, the gels stained with kumasi blue to determine the peak fractions containing V nps fractions containing the V NPS are then concentrated by centrifugation and the purified VRPs are applied to a copper TEM specimen grid, which is then negatively stained for visualization of the VRPs by transmission electron microscopy. Hi, I am WinCo Wu in the laboratory of Dr.Nellie Pane in the Department of Zoology at the University of British Columbia.
I'm Lindsay Weaver, formerly from the Pane Lab. Today we will show you a procedure for the purification of influenza, a viral rib nucleo protein complexes. We use this procedure in our laboratory to study the nuclear import of influenza, A virus in tissue culture cells.
So let's get started. To begin the experiment at 750 microliters of MNT buffer into one 11 millimeter by 34 millimeter Beckman polycarbonate centrifuge tube. Next, add 500 microliters of a two milligram per milliliter solution of influenza, A virus into the tube pipette up and down several times to mix the virus with the MNT buffer.
After mixing, place the tube in A TLA one 20.2 rotor and centrifuge in a Beckman Optima max E centrifuge for 10 minutes at 109, 000 times G at four degrees Celsius. When the spin is done, remove the supernatant, then resus, suspend the pellet in 500 microliters disruption buffer vortex. The resuspended pellet vigorously and then shake at 31 degrees Celsius for 20 minutes in an einor thermo mixer.
When the shaking is done, proceed with the glycerol gradient sediment velocity centrifugation. To prepare the glycerol gradient, place the following of glycerol into a Beckman UltraClear 13 millimeter by 51 millimeter centrifuge tube, one milliliter 70%volume by volume GLYCEROL 0.75 milliliter 50%glycerol 0.375 milliliter, 40%glycerol, and 1.8 milliliters, 33%glycerol. These glycerol solutions are made by mixing pure glycerol with NM buffer.
Also prepare a balance tube containing both glycerol and buffer. Then vortex the disrupted viral sample again and load it onto the glycerol gradient. Place the tube in a Beckman MLS 50 swinging bucket rotor and centrifuge the gradient for three hours and 45 minutes at 217, 000 times G at four degrees Celsius.
After the centrifugation is complete, manually collect 250 microliter aliquots of the gradient starting from the top of the tube. Keep fractions on ice or at four degrees Celsius. Once the fractions are collected, we are ready to analyze them using SDS poly acrylamide gel electrophoresis.
To begin the SDS page analysis, remove 20 microliters from each fraction to a fresh tube. Then add five microliters of five times SDS page sample buffer, and heat the sample to 95 degrees Celsius for five minutes After heating, spin down the samples briefly and load them onto a 10%Poly acrylamide gel include molecular weight markers in one of the wells. Next, run the samples through the gel until the brom phenol blue loading dye reaches close to the bottom of the gel.
Remove the gel from the SDS page apparatus and stain the gel with kumasi blue. Based on the staining results, identify the fractions that contain mainly influenza. A nucleo protein.
The influenza A NP is approximately 56 kilodaltons in size and is the major protein found in viral ribonucleoprotein complexes. So the NP band generally is the strongest band in the fractions containing the VRN ps. In the next part, we will concentrate these VRNP fractions.
Combine the glycerol fractions containing mainly NP, and distribute them into two Beckman 11 millimeter by 34 millimeter polycarbonate centrifuge tubes fill each tube with ethyl pyro carbonate or DEPC treated ultrapure water. Then pipette up and down several times to mix. Place the tubes in a Beckman TLA one 20.2 rotor and centrifuge for four and a half hours at 157, 000 times G at four degrees Celsius.
When the spin is complete, remove the supernatant and resuspend the pellet. In 50 microliters of DEPC treated water aliquot, the purified V NPS set aside one aliquot and store the others at negative 80 degrees Celsius. Now we are ready to proceed with the negative staining of VRPs.
Dilute one microliter of purified VRNP into nine microliters of freshly made and twice filtered PBS buffer glow discharge a copper TEM specimen grid for 30 seconds. This grid was previously coated with a Parlow Dion and carbon film. Using tweezers to hold the freshly glow discharge specimen grid, apply a five microliter drop of diluted VRNP onto the specimen grid.
Allow the drop to absorb onto the grid for eight minutes while waiting. Place a small strip of parfum in a tray, then dispense onto the parfum two drops containing 10 microliters each of PBS buffer, as well as an 80 microliter drop of the freshly made staining solution, which is composed of 1%ammonium meli date. Now wash the specimen grid by carefully lowering it into the two drops of PBS for a total time of one minute.
Then use a piece of filter paper to wick off part of the sample solution from the specimen grid without letting the specimen grid dry. Immediately after wicking off the last drop of buffer from the specimen grid, completely submerged the specimen grid within the stain droplet. And wait one minute.
After one minute, use a new piece of filter paper to completely wick off the stain solution from the specimen grid. Allow the specimen grid to air dry for several minutes before observation. Under a transmission electron microscope here are representative transmission electron microscopy images of negatively stained V nps.
As you can see here, the V NPS resemble rod-shaped particles with variable length that are approximately 30 nanometers to 120 nanometers in length. The oligomeric NP is organized as a chain of NP molecules that is further folded into a double helical repeat structure. So loops on either ends of these rod shaped particles can sometimes be seen.
We've just shown you how to purify and visualize influenza. A viral rib nucleoprotein complexes When doing this procedure. It's important not to use PBS when diluting the VR mps if you're using urinal acetate for negative staining.
So that's it. Thanks for watching and good luck with your experiments.
