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 Agro-infiltrate combinations for pull-down. Make sure to take along appropriate controls (e.
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 Agro-infiltrate combinations for pull-down. Make sure to take along appropriate controls (e.g. GFP, similar proteins expected not to bind, mutant proteins) 3dpi isolate apoplastic fluid (AF) by vacuum infiltrating leaves with ice-cold extraction buffer. Remove excess extraction buffer by dabbing gently with tissue paper.
Based on (as in copy-pasted with minor modifications from) Dyballa & Metzger, 2009. Increased the phosphoric acid concentration to 8% based on Pink et al., 2010. Staining solution:
5% Aluminium sulfate 14-18 hydrate 10% Ethanol 0.02% CBB G-250 8% Phosphoric Acid
Note: for the preparation of the staining solution, the sequential addition of the components in the following order has to be maintained: first dissolve aluminum sulfate in Milli-Q water thereafter add ethanol, homogenize, and mix CBB G-250 to the solution Let CBB dissolve properly for ~10min as soon as the solution is completely dissolved, slowly add phosphoric acid (the incorporation of the acid to the alcoholic media lets the Coomassie molecules aggregate into their colloidal state) finally fill up with Milli-Q water Store in dark bottle
Note: the final staining solution has a dark green-bluish appearance and is full of particles swimming around.