Phage stock ought to be 10111013cfu/ml. Count number colonies in plates which have many hundred or so colonies approximately. e.g., if colony amount is 50 within a dish with 100 l of 108dilution, 50 X 108X 10= 5 1010pfu/ml. == SUPPORT Process 2: ANTIGEN PREPARATION == To create GPC3 antigen for verification for GPC3 binder, pReceiver vector containing a full-length GPC3 cDNA accompanied by a His6 label at C-terminal was employed for transfection. amplification with Tomlinson scFv phage screen libraries using recombinant Rabbit Polyclonal to Caspase 9 (phospho-Thr125) individual GPC3 protein as goals (Fig. 1). We explain the phage antibodies that bind to heparan sulfate on glypicans with the panning and selection in the essential Process. In the Alternative Protocol, we describe another method of isolating binders for heparin sulfate on glypicans. The method of cloning, expression, and purification of GPC3 necessary for panning for phage is usually described in the Support Protocol 2. It also describes critical actions in troubleshooting and background information about the phage display library used here (see COMMENTARY). == Physique 1. == Flow chart for phage display. == BASIC PROTOCOL 1: PHAGE DISPLAY FOR GPC3 == This protocol Elacridar (GF120918) describes the overall methods required for phage display, including phage library growth, phage concentration by polyethylene glycol (PEG) precipitation, panning and characterization of antibodies using ELISA, FACS, and immunohistochemistry. == Materials == == For phage amplification == 2YT medium (See Reagents and Solutions) Ampicillin-100 mg/ml (Teknova) Kanamycin-50 mg/ml (Teknova) 50% w/v glucose solution (Teknova) M13KO7 helper phage (NEB) PEG 6000/NaCl Solution (Teknova) Elution buffer: 100mM HCl Trypsin (Sigma-Aldrich) 50 % v/v Glycerol (Teknova) == For ELISA == PBST (wash buffer): 1 PBS made up of 0.1% v/v Tween 20 (Sigma). Blocking solution (2 % w/v Skim milk) Dissolve 0.4 g of Difco skim milk in 20ml of 1 1 X PBS per one 96 plates, use fresh blocking buffer. MaxiSoap 96-well plates (Sigma-Aldrich). HRP-conjugated anti-M13 phage antibodies (GE Healthcare Life Sciences) Phage display library- Human single-fold scFv libraries Tomlinson I+J Elacridar (GF120918) (Medical Research Council) (see Commentary) 1M Tris-HCl (pH 8.0) E. coliF+ strain: TG1 (for phage production) and HB2151 (for soluble scFV production) were included in Human single-fold scFv libraries Tomlinson I+J. TG1 electrocompetent cells can also be purchased from Lucigen (Middleton, WI) Isopropyl -D-thiogalactopyranoside (IPTG) 0.22 m syringe filter (Millipore) LB agar plates with 100 g/ml ampicillin TMB Chromogen Solution (ThermoFisher Scientific) == For immunohistochemistry == Xylene (Sigma-Aldrich) Ethanol absolute (Sigma-Aldrich) == For HS20 IgG construction == pFUSE-CHIg-HG1 (Invivogen) for heavy chain cloning pFUSE2-CLIg-hk (Invivogen) for light chain cloning Platinum Taq DNA Polymerase High Fidelity (ThermoFisher Scientific) One Shot TOP10 Chemically CompetentE. coli(ThermoFisher Elacridar (GF120918) Scientific) 293T cells (ThermoFisher Scientific) DMEM (ThermoFisher Scientific) FreeStyle293 Expression Medium (ThermoFisher Scientific) HiTrap Protein A High Performance (GE Healthcare Life Sciences) == Gear == Bacterial shaker-incubator 50-ml conical tube or flask 96-well U-bottom microtiter plate (Sigma-Aldrich) Centrifuge with 96-well microtiter plate adapter NanoDrop (ThermoFisher Scientific) FACSCalibur (BD Biosciences) 100 mm dish 150 mm dish == Growing the libraries == Add 100 l of the library glycerol stock to 50 ml pre-warmed 2YT made up of 100 g/ml ampicillin and 1 % v/v glucose. Grow the libraries at 37C with shaking at 225 rpm until the OD 600 is usually 0.4 (~1hr). Add 50 l M13KO7helper phage (final concentration 1 108pfu/ml). Incubate for 30 minutes at 37 C without shaking, then incubate for an additional 30 minutes at 37 C with shaking at 250 rpm. Centrifuge at 3,000 g for 10 min, decant the supernatant and resuspend the TG1 cells in 100 ml of 2YT made up of 100 g/ml ampicillin, 50 g/ml kanamycin. Incubate cultures overnight at 30 C with shaking at 250 rpm. Centrifuge the overnight culture at 3,300 g for 30 min, discard pellet and filter the supernatant using 0.22 m filter. == Phage concentration by PEG precipitation == Add 12.5 ml PEG/NaCl (20 % w/v Polyethylene glycol 6000, 2.5 M NaCl) to 50 ml of the phage supernatant and mix well and leave for 1 hr on ice to precipitate phage. Centrifuge at 3,300 g for 30 min, discard the supernatant and aspirate any remaining phage supernatant carefully. Resuspend the pellet in 4 ml PBS to dissolve the phage library thoroughly and aliquot the phage library into 1 ml per vial. Store the phage Elacridar (GF120918) at 4C for short-term storage (up to a few weeks). Determine the phage titer (see Support Protocol 1) == Selection on 96-well plates == Coat maxiSoap 96-well plates overnight.