I try doing purification 146S antigen of the foot and mouth disease virus by 15-45% sucrose gradients centrifuge by Beckman SW 41 Ti rotor. However, I don't know need how much RCF (or RPM) on Beckman SW41 Ti rotor and time how long to purification ?
Please for me advice and help,
Thank you so much
Elvis
Prepare sucrose gradient 25-45% (w/w) with (0.05 M Tris, 0.1 M NaCl, 0.1% sodium azide, pH 7.6). Add 1 ml of the virus suspension carefully on top of the sucrose gradient. Centrifuge for 3.5 h at 28,000 x g using swinging bucket rotor in an ultracentrifuge. After centrifugation remove 1ml of the top and discard. The gradients were scanned at 254 nm using UA-6 absorbance detectors. Collect the peak FMD fraction. Measure the absorbance value at 254 nm. The concentration of FMD purified 146S antigen is calculated by multiplying the absorbance value by 131.55 and expressed as micro grams/ml. Check the OD value by antigen ELISA with suitable dilutions. Aliquot, seal the cryovials with parafilm and store under liquid nitrogen vapour (-196°C).
I'm not sure if SW40 and SW41 rotor diameters are same but I use 40 000 rpm and 2 hours for SW40 rotor in ultracentrifuge. Regards
at first step you can use the sucrose 20% as a cushion for prepare semi semi purify virus in 30000 RPM with SW40 beckman rotor . in this situation may be the virus purify 70- 80% and separate from other culture medium proteins . but if you want to purify the virus up to 95% you must run the sucrose density gradient with SW40 30000 RPM ( i think 28000 g as nagendarkumar said is very low and insufficient for virus Purification in SDG) for 3 hours in 8 -12°C ( higher temperature for decrease the sucrose viscosity ) . during the centrifugation the vaccume must be run and remaining till end of test .if the band is visible take the band carefully ( may be two or three band ) and read in 280and then take the sample and read in 260nm and multiplying by 131 to calculate 140s quantity .
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