CoIP

2 min read proteins

Preferred controls

  • non-interactng protein with similar properties as bait protein (non-specific binding to bait protein)
  • Expression of bait and prey seperately (unspecific binding to matrix)
  • GFP (unspecific binding to matrix and to GFP). GFP is not always the best control as it may be too abundant, and therefore anything that has even the smallest affinity for GFP will be pulled down

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 snap-freeze tissue in liquid nitrogen. Weigh. Grind tissue carefully in a pestle and mortar. Add 3 v/w of extraction buffer to the pestle and mortar. Wait for the paste to become liquid and pipet into centrifugation tubes. Let the full-leaf extract (FLE)stand for 10min to extract proteins
  3. Centrifuge FLE 20min, 40 000g, 4°C. Take liquid with as few solids as possible.
  4. Take FLE out into 1.5ml low-bind tubes, centrifuge 16,100g for 1min (to get rid of last particles)
  5. In the meanwhile take 10μl GFP-trap slurry (ChromoTek GFP-Trap) per sample and wash twice with extraction buffer at 2000g. Use a cut pippete tip
  6. Add 1ml FLE to the beads taking care not to disturb the pellet. Also collect an input sample at this stage
  7. Incubate for 30min at 4°C while rotating
  8. Wash 6 times with extraction buffer at 2000g
  9. Remove as much liquid as possible and add 50μl 4x sample buffer to the beads, boil for 10min at 95°C
  10. Run gels
  11. Blot
  12. Block 1hour room-temperature with 5% skim milk TBS-T (can also be done overnight at 4°C)
  13. Wash 3x 5min with TBS-T
  14. 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 or other appropriate antibodies (can also be done overnight at 4°C)
  15. Develop blot
  16. 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

Protein

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