Step 1: Creating a Custom Protein Array
Preparation of expression plasmids for HaloTag® bait proteins. Eight HaloTag® fusion bait proteins were selected: HaloTag®-cJun, HaloTag®-cFos, HaloTag®-PKA, HaloTag®-R1α, HaloTag®-PKA, HaloTag®-p53, HaloTag®-p65 and HaloTag® protein only. The vectors were constructed using pFN19K (N-terminal HaloTag® fusion) vectors. The HaloTag®-only negative control (pNull 19A) was made from pFN19A HaloTag® T7 SP6 Flexi® Vector by eliminating the barnase gene with the restriction digest of SgfI and PmeI (Promega), followed by a ligation of the annealed oligos (5´ OH CGCGTAAGGGTAGGTTT; 5´ OH AAACCTACCCTTACGCGAT). A detailed protocol for this construct is available here. This was followed with a transformation into high-efficiency JM109 competent cells (Promega).
Expression of proteins in cell-free TnT® T7 Quick Transcription/Translation System. Proteins were expressed in the TnT® T7 Quick System. This expression system is a coupled transcription/translation system based on rabbit reticulocyte and requires only the addition of 1.0µg of plasmid DNA for a 50µl expression reaction and incubation for 90 minutes at 30°C.
Custom Array Design. We used the HaloLink™ Array Gaskets to make 50 wells on the HaloLink™ Slides. The gasket is attached by placing the HaloLink™ Slide onto the gasket and gently pressing to make leakproof wells that eliminate well-to-well cross-contamination. Each well is 3.0mm in diameter and 1.0mm in height and has a capacity of 5–10µl. The gasket can be easily removed at the end of the assay for scanning and quantitating the signal. To capture HaloTag® fusion proteins, 5.0µl of sample is added to each well and incubated for 1.0 hour in a humidity chamber. The slide is washed with PBS containing 0.05% IGEPAL detergent (PBSI) and dried by centrifugation at
350 × g for 3 minutes. The HaloLink™ Slide containing captured proteins is then used for downstream applications.
Prey Protein Interaction
Prey proteins (c-Jun, c-Fos and p50) are cloned into TS1K vectors (non-HaloTag® analog of pFN19K) and expressed using the TnT® T7 Quick cell-free expression system. Prey proteins were labeled with biotin during expression using Transcend™ tRNA. For expression 1.0 µg of plasmid DNA is added to 50µl expression reaction and incubated for 90 minutes at 30°C.
Biotinylated prey proteins (c-Jun, c-Fos and p50) are added (5µl/well) to columns 3, 4 and 5, respectively, of the custom array. The slide is incubated for 1.0 hour, washed with PBSI and spin dried by centrifugation at 350 × g for 3 minutes. The positive protein:protein interactions are detected by incubating with AlexaFluor® 647 labeled streptavidin (10.0 µg/ml in PBSB) for 1.0 hour. All incubations were done in humidity chamber and washes used PBSI.
In parallel, columns 1 and 2 are probed by sequential 1.0-hour incubations with Anti-HaloTag® polyclonal antibody (pAb; 5µg/ml in PBSB) and anti-Rabbit IgG labeled with Alexa Fluor® 633 (10ug/ml in PBSB). The slide was scanned on Genepix® 4000B fluorescent slide scanner at 10µm resolution using 635nm Laser, PMT at 700V. For analysis, median intensity from a standard 2.0mm diameter circle from the center of each well is used.
Modulating Protein:Protein Interactions
FRB protein was inserted into the pFC20K HaloTag® Flexi® T7 SP6 Vector to create a C-terminal FRB-HaloTag® fusion protein. FRB-HaloTag® bait protein is expressed in TnT® SP6 High-Yield Wheat Germ Protein Expression System by using 8µg of plasmid DNA/50µl reaction mix and incubating for 120 minutes at 25 ºC.
The prey, FKBP, was expressed as a GST fusion in E. coli from pFV2A plasmid and purified using GST Column (final concentration 4.0mg/ml). To create a custom HaloLink™ array for HaloTag®-FRB:FKBP interactions, 5.0µl of cell free reaction containing FRB-HaloTag® protein was added to 45 wells. TnT® SP6 High-Yield wheat germ reaction without any expressed protein was added to 3 wells to serve as a negative control. The slide was incubated for 1.0 hour, washed with PBSI, spin dried and used for FKBP-GST interaction.
Twenty micrograms per milliliter of FKBP-GST protein in PBS was incubated with varying amount of rapamycin for 30 minutes to allow FKBP-Rapamycin complex to form. BSA (100mg/ml in PBS) was added to FKBP-Rapamycin solutions to a final concentration of 10mg/ml. Five microliters of solution was added in triplicate to the HaloLink™ Slide. The slide was incubated for 1.0 hour in a humidity chamber followed by washing with PBSI and spin-drying. Binding of FKBP-GST to FRB-HaloTag® at the surface was detected by sequential 1.0-hour incubations with goat anti-GST pAb followed by anti-Goat secondary Ab labeled with Alexa Fluor® 647 (10µg/ml in PBSB).
The slide was scanned at 10mm resolution on Geneix® 4000B scanner using 635nm laser and 600V PMT. For analysis, median intensity from a standard 2.0mm diameter circle from the center of each well is used.
HaloTag®-PKA fusion is expressed in the TnT® SP6 High-Yield Wheat Germ System using 8µg of plasmid DNA/50µl reaction mix and incubating for 120 minutes at 25°C. The plasmid DNA for PKA was the same as used in protein:protein interaction studies. After expression, a dilution series of cell lysate containing PKA-HaloTag® enzyme is made in PBSB. Five microliter aliquots of each dilution were added in triplicate on a HaloLink™ Glass slide. The slide was incubated for 1.0 hour in a humidity chamber, washed with PBSI containing 5% glycerol, spin-dried and used for the enzyme assay.
For the PKA assay, we used the ProFluor® PKA Assay (Promega). The protocol for the ProFluor® Assay was modified for use on HaloLink™ Slides to obtain the best signal-to-background ratio. Six microliters of Reaction buffer A containing, 50µM ATP, and 0.625µM peptide substrate was added to wells containing immobilized HaloTag®-PKA and incubated at room temperature for 1.0 hour. Control reactions were carried out in the wells with no PKA. Reactions were terminated by adding 2.0µl of termination buffer to each well and incubating for 10 minutes. The reactions were stabilized by adding 2.0µl of stabilization buffer, and fluorescence signal was measured using a Typhoon® scanner (Ex: 488nm, and Em: 520nm). Only specific scanners with the capability for reading slides filled with solution should be used for this experiment. Many conventional microarray slide scanners can only read dried slides and are not suitable for this experiment.
After reading slides on Typhoon®, the slide was washed with PBSI, dried and probed with anti-PKA antibody followed by secondary antibody labeled with Alexa Fluor® 633 (10µg/ml).