Heparin Affinity Chromatography-Based Baculovirus Purification: A Technique to Isolate Baculovirus From Insect Cell Supernatant
내레이션 대본
Set up the chromatography system by placing the wash buffer line from inlet port A1 into a bottle containing at least 500 milliliters of wash buffer. Use the A2 buffer inlet port for loading the elution buffer.
Insert several 50-milliliter conical tubes into the fraction collector to collect the column pass-through baculovirus supernatant, the wash buffer, and the eluted baculovirus. Equilibrate the heparin column with five 7.9-milliliter column volumes of wash buffer, at a linear flow rate of 7 milliliters per minute.
Load 250 milliliters of baculovirus supernatant onto the heparin column, using the sample pump of the inlet sample port at a linear flow rate of 2 milliliters per minute. Then, run 10 column volumes of wash buffer through the heparin column at a linear flow rate of 2 milliliters per minute, until the ultraviolet absorbance curve has returned to baseline and become stable.
Dilute the baculovirus particles from the heparin column with five column volumes of elution buffer at a linear flow rate of 4 milliliters per minute. Watch for a sharp elution peak of protein on the chromatogram when the baculovirus particles dissociate from the heparin column.
Post-elution, immediately dilute the eluted baculovirus supernatant 10-fold using 20 millimolar sodium phosphate buffer to prevent inactivation of baculovirus particles from osmotic shock during subsequent centrifugation.
