Bimolecular Complementation Affinity Purification to Isolate Two Interacting Proteins

1. Cell Culture and Transfection

NOTE: For transfection of the BiFC vectors it is important to achieve a high efficiency and relatively homogenous transfection. The vectors will likely be compatible with any standard transfection reagent, and the transfection conditions should be optimized accordingly. To perform mass spectrometry, we usually culture cells within 10 cm dishes, although this can also be proportionally scaled down to smaller dishes or plates for experiments that require less material.

1. Seed 1.0 × 10⁶ HEK293T cells in a 10 cm dish with 10 mL of DMEM media, supplemented with 10% FBS and Penicillin/Streptomycin (1:100).
2. Dilute 2.5 µg of each BiFC vector into 500 µL of transfection buffer (see Table of Materials) in a 1.5 mL microcentrifuge tube.
3. Add 10 µL of transfection reagent (see Table of Materials).
4. Vortex mixture for 10 s, then briefly centrifuge. Incubate for 10 min at room temperature.
5. Add the DNA transfection mixture to the plate, dropwise. Incubate the cells for a sufficient length of time for the BiFC fusion proteins to interact and for Venus folding and fluorophore maturation, generally ~8 – 24 h.
   NOTE: The peak excitation for Venus is 515 nm, and its peak emission is 528 nm, although this is readily viewed using a standard fluorescent microscope set up to visualize GFP fluorescence.

2. Sample Preparation

1. Harvesting lysates
   1. Prior to lysate collection, prepare Cell Lysis Buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% (v/v) non-ionic detergent (see Table of Materials)]. Store at 4 °C.
   2. Immediately prior to harvesting, supplement 10 mL of Cell Lysis Buffer with protease inhibitor and phosphatase inhibitor (see Table of Materials). Keep supplemented Cell Lysis Buffer on ice.
   3. Wash cells twice in ice-cold PBS. Aspirate the PBS and add 1 mL of ice-cold supplemented Cell Lysis Buffer. Place the dish on ice, ensuring the buffer is spread over the entire surface area.
   4. Incubate on ice for approximately 5 min, then use a cell scraper (see Table of Materials) to scrape the cells and transfer them to a pre-chilled 1.5 mL microcentrifuge tube.
   5. Remove cellular debris by centrifuging the collected lysate at 18,000 × g for 5 min at 4 °C and transfer cleared supernatant into fresh microcentrifuge tubes.
      NOTE: At this point, the lysate can be used immediately, or stored at -80 °C. It is also recommended to keep an aliquot of crude lysate so that the efficiency of transfection and affinity purification can be validated.
2. Affinity purification
   NOTE: The BiCAP isolation step is performed using the GFP nanobody conjugated to agarose beads (see Table of Materials).
   1. Prepare the agarose beads by washing an appropriate volume (20 µL per sample + 10 µL excess) in 1 mL PBS. Centrifuge the beads at 300 x g and remove the supernatant.
   2. Add 20 µL to each lysate sample.
   3. Incubate the samples for 2 h at 4 °C with end-to-end rotation.
      NOTE: At this point, the samples can be prepared for either SDS-PAGE and western blotting, or analysis by mass spectrometry.
3. Preparation of BiCAP eluent for western blotting.
   1. Centrifuge the beads at 300 x g and wash them three times in the Cell Lysis Buffer.
   2. Resuspend the washed beads in 50 µL of appropriately diluted sample buffer (see Table of Materials) and heat the samples at 95 °C for 2-3 min.
      NOTE: Samples prepared in this manner can be stored at -20 °C for several months.
   3. Perform SDS-PAGE and western blotting for both the V1 tag and V2 tags (see Table of Materials), as well as any other proteins of interest.