Figure 1. Protein fragments of Venus (super enhanced YFP) constructs. A. Venus protein (amino acids 1-239; accession number: CAO79509) was dissected into two fragments at residue 158 to generate Venus N-terminus (top) and Venus C-terminus (bottom). B. Schematic drawing of Venus-N-p53 and Venus-C-PDID fusion fragments. Venus-N-p53 and Venus-C-PDID contain Venus-N-terminus and Venus-C-terminus fused respectively to p53 (amino acids 1-393; Gully et al., 2012) and the phosphorylation-dependent interaction domain (PDID, amino acids 287-530) of human Brd4 (Wu et al., 2013), in which a flexible linker containing two copies of Gly₄Ser peptide is introduced to allow optimal space contacts between Venus-N-terminus and Venus-C-terminus and also to prevent steric hindrance between the Venus fragment and its fused protein of interest. AscI and XbaI indicate the positions of restriction enzyme-cutting sites used for generating fusions from PCR-amplified DNA fragments. An initiation codon for methionine (M) was added to allow translation of Venus-C-PDID. It should be noted that, although linker peptides ranging from 5 to 17 amino acids are often used (Remy and Michnick, 2007), the exact length and the sequence nature of the linkers have not been systematically analyzed (Kerppola, 2013). C. BiFC fluorescence signal is produced when Venus-N and Venus-C are in close proximity brought together via p53-PDID interaction in the cell.

Materials and Reagents

Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F2442 )
Antibiotics (Penicillin/Streptomycin) (Sigma-Aldrich, catalog number: P0781 )
Cell culture medium (Complete: With 10% FBS and antibiotics; Antibiotic-free: with 10% FBS only)
Formaldehyde (Thermo Fisher Scientific, catalog number: F79-500 )
Triton X-100 (Sigma-Aldrich, catalog number: 79284 )
BSA (Sigma-Aldrich, catalog number: A3059 )
Lipofectamine 2000 (Life Technologies, Invitrogen™, catalog number: 11668-019 )
Venus-N-p53 (Gully et al., 2012) (Santa Cruz, catalog number: sc8334 ) and Venus-C-PDID (Wu et al., 2013) plasmids (Santa Cruz, catalog number: sc5384 ) (see Figure 1B)
Primary antibodies against Venus
e.g. Anti-full-length-GFP antibody (Santa Cruz, catalog number: sc8334 or sc9996 )
Anti-C-terminal GFP antibody (Santa Cruz, catalog number: sc5384)
or β-actin (Sigma-Aldrich, catalog number: A5441 )
Secondary antibody conjugated with a fluorescence dye emitting wavelength other than that of Venus (excitation 488 nm, emission 515 ± 15 nm) or Hoechst 33258 (excitation 350 nm, emission 461 nm), for example, Alexa Fluor^® from Life Technologies
Sodium chloride (Thermo Fisher Scientific, catalog number: 7647-14-5 )
Potassium chloride (Thermo Fisher Scientific, catalog number: 7447-40-7 )
Sodium phosphate dibasic heptahydrate (Na₂HPO₄^.7H₂O) (Thermo Fisher Scientific, catalog number: S373500 )
Potassium phosphate monobasic (KH₂PO₄) (Thermo Fisher Scientific, catalog number: 7778-77-0 )
Aluminum foil (grocery store)
Permanent mounting medium (Vector Laboratories, catalog number: H-5000 )
Microscope slides (Thermo Fisher Scientific, catalog number: 12-544-7 )
Nail polish (grocery store)
10x Phosphate Buffered Saline (PBS) (see Recipes)
3.7% Formaldehyde (freshly prepared) (see Recipes)
Phosphate Buffered Saline with Triton X-100 and BSA (PBSTB) (see Recipes)
Hoechst 33258 (Sigma-Aldrich, catalog number: 861405 ) (see Recipes)

Equipment

Glass bottom culture dish (35-mm glass bottom plate containing a 14-mm center microwell, poly-D-lysine coated) (MatTek, catalog number: P35GC-1.5-14-C )
Tissue culture hood (NuAire, model: Class II, Type A2 )
37 °C cell culture incubator (Thermo Fisher Scientific, Forma^®, model: Series II , water-jacketed and HEPA filtered)
Confocal fluorescence microscope (Nikon, model: Eclipse TE-2000E/C1 )
Rocker (Labnet International, model: Rocker 25 )

Software

NIS Elements Basic Research (version 2.2)
Nikon EZ-C1 Free Viewer (version 3.90)

Procedure