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Selection of Recombinant Antibodies 249fill 10 11 –10 12 amplified phage solved in 150 µL panning block from the formerpanning round in the blocked wells. Incubate at RT for 2 h for binding of the antibodyphage.3. Remove the unspecifically bound antibody phage by stringent washing.Thereafter, wash the wells 10 times with an ELISA washer in the first panninground. In the following panning rounds increase the washing steps (20 times in thesecond panning round, 30 times in the third panning round, and so on.) (see Note 3).4. Elute with 200 µL trypsin solution for 30 min at 37°C (see Note 4).5. Use 10 µL of the eluted phage for titering (see titering).6. Inoculate 50 mL 2X TY-T with an overnight culture of E. coli XL1-blue MRF′(Stratagene, Amsterdam, Netherland) in 100-mL Erlenmeyer flasks and grow at250 rpm and 37°C.7. Infect exponentially (OD 600~0.5 nm, after 2–3 h) growing 20 mL XL1-blue MRF′culture with the remaining 190 µL of the eluted phage. Incubate 30 min at 37°Cwithout shaking and the following 30 min with 250 rpm.8. Harvest the infected bacteria by centrifugation for 10 min at 3200g in 50-mL PPtubes. Resolve the pellet in 250 µL SOB-GA and plate the bacteria suspension onSOB-GA agar plates (15-cm Petri dish). Grow overnight at 37°C (see Note 5).9. Harvest the grown colonies by suspending in 2.5 mL 2X TY-GA with aDrigalsky spatula.10. Use 100 µL of the harvested bacteria for the amplification of the eluted phage (seeSubheading 3.3.).11. Make a glycerin stock of the panning round by adding 250 µL 87% glycerin to750 mL of the harvested bacteria. Mix and store at −80°C.3.3. Packaging of Phagemids1. For the next panning round the eluted phage must be packaged and reamplified.Inoculate 50 mL 2X TY-GA in a 100-mL Erlenmeyer flask with 100 µL harvestedbacteria (OD 600< 0.1 nm). Grow at 250 rpm at 37°C up to an OD 600approx 0.5 nm.2. Infect 5 mL bacteria culture (~2.5 × 10 9 cells) with 5 × 10 10 PFU (multiplicity ofinfection = 1:20) of the helperphage M13K07. Incubate for 30 min without shakingand the following 30 min with 250 rpm at 37°C.3. To remove the glucose, harvest the cells by centrifugation for 10 min at 3200g in50-mL PP tubes.4. Resuspend the pellet in 30 mL 2X TY-AK in a 100-mL Erlenmeyer flask. Producethe phage for 16 h at 250 rpm and 30°C5. Pellet the bacteria by centrifugation for 10 min at 3200g in 50-mL PP tubes. If thesupernatant is not clear, centrifuge again to remove remaining bacteria.6. Precipitate the phage in the supernatant by adding one-fifth volume PEG solutionin 50-mL PP tubes. Incubate for 1 h at 4°C with gentle shaking.7. Pellet the phage by centrifugation for 1 h at 3200g and 4°C. Put the open tubesupside down on tissue paper and let the viscous PEG solution move out completely.Resuspend the phage pellet in 500 µL phage dilution buffer. Titer the phage preparationand use it for the next panning round. Store the remaining phage at 4°C.