Phage elution

Infect 1-mL culture of HB2151 with 10 pL (about 105 tu.) of the phage eluted and neutralized at the last round of selection. Plate 0.1, 1, 10, and 100 pL on TYE-AMP-GLU. Incubate overnight. 

Pellet the tissue by spinning at 6,000rpm (6,500 x g) for 3 min. Titer supernatant (as described in Subheading 3.2) to define the amount of phage eluted from the tissue (i.e., IU of phage per gram of tissue). 

The phage display library is now ready for use in an affinity selection protocol. Affinity selection has also been successfully utilized in vitro and ex vivo. The affinity selection principle is the same for all types of selection. First, allow the phage library to bind to the presented target. Wash away unbound phage, elute and amplify bound phage. Repeat the selection process three to four times. For example in vitro targets may be purified, biotinylated, and immobilized on a streptavidin-coated plate for affinity selection (9). 

On column infection of co//cells with bound phages Phage amplification Phage elution... 

Elute the bound phage by adding 700 pL of phage elution solution and rocking gently for <10 min. Transfer the eluted solution to microfiige tube and neutralize with 105 pL of neutralization solution. 

A variation on the theme has been to map out protease specificity.28 A library of fusion proteins was constructed in a modular manner. The synthetic protein had an N-terminal domain that binds very tightly to an affinity column. This domain was connected to the C-terminal domain of M13 gene III by a randomized peptide sequence. The phages were then bound to the affinity support and treated with a protease. Phages that had a protease-susceptible site were cleaved from the support and eluted. This procedure was subsequently used to map out the specificity of furin,29 which is described in the next chapter. 

Replate the eluted recombinant phage at a density of approx 500-1000 PFU/mL and rescreen with the antibody probe. Repeat the procedure of picking a positive plaque, eluting titrating, and replating at lower plaque densities until it is possible to pick a single well-isolated plaque. 

Pool the 4 x 100 pL aliquots of eluted phage, and use them to infect 2 mL of a fresh overnight culture of XLl-Blue grown in SB medium for 15 min at room temperature... 

Shake out excess PBS from the tube, and elute the bound phage by adding 1 mL 100 mM triethylamine. 

Elute the phage from the beads by resuspending in 300 pL PBS, containing 50 voMdithiothreitol (see Note 7). 

Addition to the Immunotube of 200 pL of lMTris-HCl, pH 7.4, followed by 4 mL TGI culture and then incubation for 30 min at 37°C for infection allows recovery of some phage that may have not been eluted by triethylamine. In general, however, this precaution is not required. 

In this strategy, phages are affinity-captured on immobilized substrate under conditions in which the enzyme is inactive, for example, in the absence of an essential metal ion or a cofactor. Active phage-enzymes are then eluted by triggering catalysis by addition of the metal ion or cofactor, taking advantage of the lower affinity for the product than for the substrate. 

Wash thoroughly 10 times with TBST. Elute bound phage for 5-10 min with 400 pL of elution buffer. Wash the plate surface thoroughly during this elution using a pipet tip to remove all phage. Add 75 pL of 1 M Tris-HCl, pH 9.1, and mix (see Notes 3 and 4). 

Amplification and renormalization Amplification and renormalization are combined in the simplified form used in SELEXION. Each phage surviving elution is scaled the same amount, yielding a renormalized library of the same size as the original library, but with the mole fractions that resulted from elution. 

Sampling The results of an experiment are determined by the small number of phage (typically 20-200) that are sampled from the eluted phage. Because this sample is such a minuscule fraction of the library, we include a probabilistic model of sampling in the model. 

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