Development of Arabidopsis Protein Chip

Protein Expression in N. benthamiana

  1. 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).
  2. Transfer cultures in 15 ml Falcon tubes. Spin down cultures at 3000 rpm for 20-30 min in the table top centrifuge. Discard supernatant.
  3. Re-suspend pellet in 10 ml Infiltration medium. Incubate at room temperature for at least 3 hours (it can go also overnight).
  4. 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.
  5. 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.
  6. Mix cultures with p19 in a 1:1 ratio. Make ~ 20ml for each construct.
  7. 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.
  8. After 5 days collect the leaves and freeze them in liquid nitrogen immediately. Proceed with protein extraction.

Infiltration Medium:

10 mM MgCl2
10 mM MES
200 µM acetosyringone