3.1. Protein extraction from plants and Immunoprecipitation

Pre-chill down the material to be used and keep everything on ice or in the cold room (see Note 6).

Grind tissues in liquid nitrogen (see Note 7) and transfer with a pre-chilled spatula to 2 mL of ice-cold sucrose buffer completed with the desired inhibitors according to the post-translational modification analyzed. It is also possible to use fresh tissues and grind it directly in ice-cold sucrose buffer (see Note 7).

Centrifuge for 10 minutes at 5,000 g at 4 °C to remove cellular debris and transfer the supernatant into 2 mL Eppendorf tubes.

Repeat step 2 until the supernatant is clean (see Note 8).

Pre-heat a heat block to 95°C.

Pipet 50 µL of the total protein fraction to use as input of the immunoprecipitation, and add 15 µL of 4X Laemmli buffer.

Boil the input for 3 minutes at 95°C. Keep the input on ice if you are planning to proceed with western blot the same day or freeze in liquid nitrogen and keep at -80°C for later use.

In order to obtain the membrane protein fraction, centrifuge for 45 minutes at 20,000 g at 4°C and keep the pellet (microsomal fraction) (see Note 9).

Add 1 mL of lysis buffer to the pellet (see Note 10) and resuspend carefully by pipetting up and down (see Note 11).

Transfer the lysate to ice-cold potters and homogenize (see Note 12). Transfer the samples to new 1.5 mL tubes.

Solubilize the microsomal fraction on a rotating wheel at 4°C for 30 minutes (Figure 1).

Centrifuge the samples for 10 minutes at 20,000 g at 4°C to pellet non-solubilized microsomes. Transfer the supernatant into a new 1.5 mL tube. This fraction corresponds to solubilized microsomal proteins and will be used for BRI1 immunoprecipitation.

Quantify the samples and use 1 mg of proteins of the supernatant to perform the immunoprecipitation of BRI1.

Add 50 µL of Anti-Tag MicroBeads to the supernatant and mix well (Figure 1).

Incubate on ice for 30 minutes to allow the formation of the BRI1-tag-antibody complex.

Place the µColumn on the µMACS™ Separator and apply 200 µL of the Lysis buffer to equilibrate the column (see Note 10).

Pipette 75 µL of Elution buffer for each sample into a new 1.5 mL tube and boil it on the pre-heated 95°C block.

Apply the samples lysate onto the center of the column and let it run through by gravity flow.

Wash the column with 200 µL of the Wash buffer 1. Repeat 4 times.

Wash the column with 100 µL of the Wash buffer 2.

Apply 20 µL of Elution buffer kept at 95°C onto the center of the column and incubate at room temperature for 5 minutes (see Note 13).

Place a new 1.5 mL collecting tube under the column.

Apply 50 µL of Elution buffer kept at 95°C onto the center of the column and collect the eluates (see Note 14). Freeze samples in liquid nitrogen and keep them at -80°C, or proceed directly with immunoblotting.