Hi all,
I am currently working on purifying influenza viruses. After having taken the splitting step I am now facing some problems in the Diafiltration step.
My aim in diafiltration is to replace Sucrose (remained from gradient ultracentrifuge step) and Triton (remained from splitting step) in allantoic fluid (our propagation is in embryonated eggs) with PBS.
I use Cogent M1 (Millipore) UF system with a pellicon 2 cassette (300 kD pore size, PES). Our numbers of diavolumes are 20-30 times.
My problem is that I am losing a high percentage of proteins with the Diafiltration waste. What do you think I should do?
I really appreciate if anyone could give me some advice regarding this matter.
Many thanks,
Sara
I recommend a Step by Step diafiltration. Add few volume of sucrose and triton in PBS buffer.
If possible, add Tween 20 detergent at 1/10,000 dilution in the sample and dialysis buffer.
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