TITLE: Concentrating Viruses by Tangential Flow Filtration
AUTHORS: Steven Wilhelm, Samantha Coy

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Ultracentrifugation in the WX Ultra Series can support only 30 mL samples at a time, and therefore do not significantly concentrate viruses. Tangential flow filtration enables viral concentration of large volumes that can then be purified using sucrose density gradients. </div><div class = "text-block">Contact Dr. Steven Wilhelm (wilhelm@utk.edu) or Samantha Coy (srose16@vols.utk.edu) for additional information regarding this protocol.</div></div>

[STEPS]
?. [Machine Preparation]
Set up the tangential flow filtration (TFF) apparatus so that the Pellicon XL 30 kDa filter is attached. Both the retenate and waste tubes should empty into your waste flask.*Catalog number of Pellicon XL 30 kDA filter: PXC030C50*Refer to Figure 1 in the attached PDF.
?. [Machine Preparation]
Make 500 mL 0.1 N NaOH, 45ºC. Pour into the TFF reservoir.
?. [Machine Preparation]
Turn on the pump speed to 1 or 2, and gradually turn the back pressure valve clockwise to increase the pressure. The bottom pressure gauge should be 30-40 psi, and the top pressure gauge should be ~10 psi.
?. [Machine Preparation]
Circulate the fluids until ~350 mL is left. Re-attach the retenate tube to circulate the remaining cleaning solution that does not pass through the filter. Run the NaOH solution through system for ~20 minutes.* Do NOT let the system run dry!
?. [Machine Preparation]
When 50 mL is left, turn the pump speed to off and tighten a plastic clamp onto the tubing that flows to the pump inlet. Detach the tube, and undo the clamp to empty the remaining NaOH solution into the waste vial.*Refer to Figure 2 in the attached PDF.
?. [Machine Preparation]
Attach a syringe to the tube connecting the pump outlet, and pull back the plunger to draw out any remaining NaOH inside of the machine. Re-attach the tubing, and use the syringe to drain any other tubing in the same manner (e.g. over the surface of the filter).
?. [Machine Preparation]
Wash the TFF system with at least 1L of MilliQ water, allowing the first 500 mL to empty into the flask through both the retenate and waste tubes. Re-attach the retenate tube to circulate the last 500 mL and run it through the apparatus for ~10 min. Empty the remaining volume in the same process outlined in step 5 and 6.
?. [Machine Preparation]
Circulate 300 mL of 50mM Tris-HCl, pH=7.8, 10 mM MgCl2 until ~25 mL is left.*This step primes the TFF system with buffer to protect the viruses against any residual effects of the NaOH.*Use any buffer that is appropriate for the viruses that you are concentrating.
?. [Concentration]
Fill the reservoir with the virus stock. Continue to run the system at the same psi (30-40 psi for the bottom pressue and ~10 psi for the top pressure gauge). After the sample has concentrated, empty the remaining volume in the reservior into a Falcon tube. Virus may get stuck to the filter so use a syringe attached to the pump outlet tube, and draw out the remaining fluid from the filter. It may be necessary to go back and fourth with the plunger to dislodge viruses that are stuck to the surface.
?. [Purification]
Purify the samples (e.g. filter sterilize if axenic cultures, perform sucrose density gradient if not).
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?. [Titering]
Titer the sample by plaque assay and determine the actual concentration compared to the theoretical.*Theoretical concentration = (V1*C1) = (V2*C2)
?. [Machine Cleaning]
Clean the TFF system with NaOH in the same process as in the preparation steps. Rinse with MilliQ.
?. [Machine Cleaning]
Using a syringe filter, fill the Pellicon filter with 0.1 N NaOH, 4°C until the next use.