Protein Expression in N. benthamiana
- Grow 10ml GV2260 Agrobacterium cultures containing the cDNAs wished to be expressed, in appropriate selection medium (LB with Rif25 Carb50 Strep100 Spec100), for 36h to OD600 ~1.0-1.2. Don’t forget to start a culture with p19 containing Agro strain (LB with Rif25 Carb50 Strep50 Kan50).
- Transfer cultures in 15 ml Falcon tubes. Spin down cultures at 3000 rpm for 20-30 min in the table top centrifuge. Discard supernatant.
- Re-suspend pellet in 10 ml Infiltration medium. Incubate at room temperature for at least 3 hours (it can go also overnight).
- Optional: After 3 hours at least, spin down at 3000 rpm for 15-20 min. and discard supernatants. Re-suspend in 5-10 ml of fresh Infiltration medium.
- Read the O.D.600 and adjust at 0.4-0.5 using Infiltration medium. Similarly, the p19 culture should be brought up to O.D.600~0.8-1.0.
- Mix cultures with p19 in a 1:1 ratio. Make ~ 20ml for each construct.
- Infiltrate as many leaves as you can with the Agro mixture. I usually infiltrate 3-4 large leaves starting from the top of the plant. Start with 2-3 week-old healthy plants grown in medium-sized pots. For infiltration I use 1 or 2 ml syringes without needles. Once you start infiltrating a leaf, infiltrate entirely, don’t leave empty areas, and mark it so you’ll know to collect it.
- After 5 days collect the leaves and freeze them in liquid nitrogen immediately. Proceed with protein extraction.
10 mM MgCl2
10 mM MES
200 µM acetosyringone