Motor Protein Purification from Sf9 cells
- Sf9 cells (being in low passage, log-phase growth, excellent health and more than 95% viability)
- P2 virus (being fresh and high titer)
- Lonza medium (serum free)
- Erlenmeyer flask (sterilized, 1L)
- Cells are collected at 500g for 10min, at 25°C. (The volume of cell pellet is about 5ml)
- Resuspend the cells with 1.5-fold volume of Lysis Buffer already containing 1 tablet cocktail (Roche), 1mM PMSF and 10mM β-Me. The total resuspended volume of cells is about 15ml. (Note: We add 10ml Lysis Buffer containing 150uL PMSF and 10μl β-Me for 440ml cells or 14μl β-Me for 400ml cells, in order to reach to 1mM and 10mM final concentration respectively. 1 tablet cocktail for less than 50ml solution)
- Froze the cells in liquid nitrogen. Then rapidly thaw the cells at 37°C to lyse the cells in a very short time. Repeat the above froze-and-thaw step and add fresh 1mM PMSF to the lysate. (Note: Try to avoid bubbles when thawing the cells).
- Centrifuge the lysate at 44000g for 50min, at 4°C.
- Collect the supernatant and remove the pellet. Incubate the supernatant with Ni-beads (about 1ml) for a short time (about 5-20min).
- Loaded the lysate into a column. Then flow through it.
- Wash the column with 60ml Wash Buffer 1.
- Wash the column with 20ml Wash Buffer 2.
- Elute the protein with 6ml Elute Buffer. Add 2ml buffer per time and resuspend the beads. (Note: When eluting protein from Ni beads, you need Elute Buffer more then 5-fold and less than 10-fold volume of beads)
- Concentrate the protein by centrifugation at 3000g for 30min, 4°C. Then pipet the protein solution to avoid high concentration in the bottom. When the volume is nearly 700μl, add 300μl Storage Buffer to replace the Elute Buffer gently. Centrifuge for 15min, 4°C. Repeat the above step for five times until the volume is about 500μl. (Note: Try to avoid bubbles)
- Store the protein at -80°C.
- When the cell density is near to 2×106 cell/ml, sf9 cells are at log phase, which is important for virus preparation, virus amplification and protein purification.
- when we purify kif5b, the virus-adding ratio is 1/100 (V/V).
- We collect the cells after 60-hrs infecting the cells.
- Cells (about 440ml) infected by virus K2: After eluting with 6ml Elute Buffer, the protein concentration is 0.409mg/ml. After concentrating, the protein concentration is 2.91mg/ml. (About more than 1.7mg totally)
- Cells (about 400ml) infected by virus K3: After eluting with 6ml Elute Buffer, the protein concentration is 0.331mg/ml. After concentrating, protein concentration is 3.576mg/ml. (About more than 2.5mg totally)
- We usually stop the process of concentration of kif5b when we estimate the protein concentration is about 2mg/ml.