Editing Expression of HA NA using Baculovoirus (section)

=== <br> Harvesting recombinant protein from High Five cell expression supernatants ===

<br> 1. 72-96 hr post infection transfer culture supernatants into 500 ml centrifugation buckets. Spin at 5,500 x g for 20 min at 4 °C. In the meantime rinse the shaker flasks 3x with double distilled water (ddH2O).

<br> 2. Transfer 3 ml Ni-resin slurry into a 50 ml tube with 45 ml of PBS (one needed per 200 ml culture). Shake well and spin at 3,000 x g at room temperature for 10 min. Decant the PBS and keep the pellet Mix the insect cell supernatant with the Ni-resin pellet and transfer into the already rinsed shaker flasks. Incubate at 4 °C for 2-4 hr, while shaking.

<br> 3. Mount 10 ml polypropylene column on a clamp on a support stand. Remove both caps and place a beaker below the column to collect flow through (waste). Take the shaker flask out of the shaker and start to transfer the supernatant/slurry mixture onto the column; it will retain the Ni-resin and only the supernatant will flow through. Pass the entire volume over the column.

<br> 4. Wash the resin 4x with 15 ml of washing buffer (50 mM Na2HCO3, 300 mM NaCl, 20 mM imidazole, pH 8). Let all the washing buffer drain from the column and close it with the cap (lower side).

<br> 5. Add 2 ml of elution buffer (50 mM Na2HCO3, 300 mM NaCl, 300 mM imidazole, pH 8) and let sit for 5 min. Open column and collect the eluate. Repeat this 3 more times (with a total of 8 ml of elution buffer). Eluate that contains high concentrations of protein typically shows foam when shaken. Keep eluate on wet ice whenever possible.