Using Phage Display to Select Proteins with High Affinity to a Target Protein
Published: June 29, 2023
Abstract
Source: Zhang, W., et al., Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases. J. Vis. Exp. (2021)
In this video, we demonstrate the phage display technique to isolate pre-engineered phages expressing proteins with high affinity to the target protein.
Protocol
1. Prepare the phage library Thaw the phage library on ice and dilute it to 100x the library diversity in PBS. For example, if the library diversity is 1 x 1010 and the library concentration is 1 x 1013, dilute the library so that the concentration is 1 x 1012. For the libraries used here, dilute them 10-fold in PBS by combining 1 mL of the library with 9 mL of PBS. NOTE: Library diversity should have been determined during library creation and cannot b…
Divulgations
The authors have nothing to disclose.
Materials
| Axygen Mini Tube System (0.65 mL, sterile, 96/Rack, 10 Racks/pack) | Fisher Scientific | 14-222-198 | Plastic ware |
| BD Difco Dehydrated Culture Media: LB Broth, Miller (Luria-Bertani) | Fisher Scientific | DF0446-17-3 | Preparing plates for titering |
| Bovine Serum Albumin (BSA), Fraction V | BioShop Canada | ALB001 | Buffer component |
| Carbenicillin disodium salt 89.0-100.5% anhydrous | Millipore-Sigma | C1389-5G | Culturing phagemid-infected cells |
| Compact Digital Microplate Shaker | Fisher Scientific | 11-676-337 | Device |
| Corning Microplate Aluminum Sealing Tape | Fisher Scientific | 07-200-684 | Sealing phage glycerol stocks |
| Dehydrated Agar | Fisher Scientific | DF0140-01-0 | Preparing plates for titering |
| DS-11 Spectrophotometer/Fluorometer | DeNovix | DS-11 FX+ | Device |
| Greiner Bio-One CellStar 96-Well, Non-Treated, U-Shaped-Bottom Microplate | Fisher Scientific | 7000133 | Plastic ware |
| Hydrochloric Acid | Fisher Scientific | A144-500 | Chemical |
| Invitrogen One Shot OmniMAX 2 T1R Chemically Competent E. coli | Fisher Scientific | C854003 | Bacterial strain for phage infection |
| Kanamycin Sulfate | Fisher Scientific | AAJ1792406 | Culturing M13K07 helper phage-infected cells |
| M13KO7 Helper Phage | New England Biolabs | N0315S | Permit phagemid packing and secretion |
| MaxQ 4000 Benchtop Orbital Shaker | Fisher Scientific | 11-676-076 | Device |
| Nunc MaxiSorp 96-well microplate, flat bottom | Life Technologies | 44-2404-21 | Plastic ware |
| Phosphate-Buffered Saline (PBS) 10X Solution | Fisher Scientific | BP3994 | Buffer component/phage resuspension medium |
| Polyester Films for ELISA and Incubation | VWR | 60941-120 | Covering the microplates during incubation |
| Polyethylene Glycol 8000 (PEG) | Fisher Scientific | BP233-1 | Phage precipitation |
| Sodium chloride | Millipore-Sigma | S3014 | Phage precipitation |
| Sterile Plastic Culture Tubes: Translucent Polypropylene | Fisher Scientific | 14-956-1D | Culturing phage inputs |
| Tetracycline Hydrochloride | Fisher Scientific | BP912-100 | Culturing E. coli OmniMax cells |
| Tris Base | Fisher Scientific | BP1525 | Neutralizing eluted phage solution |
| Tryptone Powder | Fisher Scientific | BP1421-2 | Cell growth media component |
| Tween 20 | Fisher Scientific | BP337500 | Buffer component |
| Yeast Extract | Fisher Scientific | BP1422-2 | Cell growth media component |
Tags
Citer Cet Article
Using Phage Display to Select Proteins with High Affinity to a Target Protein. J. Vis. Exp. (Pending Publication), e21408, doi: (2023).