Preferred controls

• GFP (unspecific binding to matrix and to GFP)
• non-interactng protein similar to bait (non-specific binding to bait)
• No GFP-containing protein (unspecific binding to matrix)

Infiltration

1. Agro-infiltrate combinations for pull-down. Make sure to take along appropriate controls (e.g. GFP, similar proteins expected not to bind, mutant proteins)
2. 3dpi isolate apoplastic fluid (AF) by vacuum infiltrating leaves with ice-cold extraction buffer. Remove excess extraction buffer by dabbing gently with tissue paper. fold leaves into a 20ml syringe in a 50ml tube and centrifuge 25min 4°C 2000g, slow accelaration and deceleration.
3. Take AF out into 1.5ml low-bind tubes, centrifuge 16,100g for 5min (to get rid of Agrobacterium and other debris)
4. In the meanwhile take 10μl GFP-trap slurry (ChromoTek GFP-Trap) per sample and was twice with extraction buffer at 2000g. Use a cut pippete tip
5. Take 1ml AF to the beads and add 2μl 500mM PMSF (final 1mM) and 1μl protease inhibitor cocktail (0.1%(v/v)
6. Incubate for 30min at 4°C
7. Wash 6 times with extraction buffer at 2000g
8. Remove as much liquid as possible and add 50μl 4x sample buffer to the beads, boil for 10min at 95°C
9. Run gels
10. Blot
11. Block 1hour room-temperature with 5% skim milk TBS-T (can also be done overnight at 4°C)
12. Wash 3x 5min with TBS-T
13. Incubate the membrane with 1:5000 diluted anti-GFP (Abmart, Berkeley Heights, NJ, USA, M20004L), anti-HIS 1:5000 (Abmart, Berkeley Heights, NJ, USA, M20003L) antibodies in 5% milk TBS-T (can also be done overnight at 4°C)
14. Develop blot
15. Stain blot with CBB

Our specifics

• 14% SDS-PAGE for prey (aHIS)
• 10% SDS-PAGE for bait (aGFP)

Buffers

Extraction buffer:

• 200mM NaCl
• 50mM MOPS pH 7.5 (set with NaOH)
• 10 mM ethylenediaminetetraacetic acid
• 1.0% (v/v) NP-40 (Igepal CA-630)

Add to extraction buffer before use

• 1mM phenylmethylsulfonyl fluoride (500mM stock in DMSO)
• 0.1% (v/v) protease inhibitor cocktail (P9599;Sigma, St. Louis, MI, USA)