Figure 3.

Synchronous selection of a random peptide iPhage library. KS1767 cells are incubated with the random peptide iPhage library for 24hr at 37°C. The following day, cells are washed with PBS and subsequently detached with trypsin. Cells are incubated with hypotonic buffer, and placed in a standard Dounce homogenizer to disrupt cell membranes. The mitochondria/endoplasmic reticulum (ER) fraction is obtained by differential centrifugation at 4°C. The subcellular fraction-bound phage population is recovered through infection of log-phase K91/kan E. coli. Phage is purified by PEG/NaCl precipitation and prepared for the second round of selection. This process is performed as many times as needed. Usually two to four rounds of selection are enough to isolate enriched iPhage particles.