Cell-specific labeling by a synthetic peptide derived from phage containing a PMN-binding peptide sequence. Suspensions of 1 × 107 HT29 epithelial cells (A) or PMN (B) in HBSS containing 0.5% BSA were incubated at 4°C for 1 hour in the presence (+) or absence (−) of 1 mmol/L FGPNLTGRW followed by the addition of Biotin-GF(Benzoyl)GPNLTGRW to a concentration of 100 μmol/L. After a 30-minute incubation in the dark, the samples were placed on ice and photolyzed for 10 minutes under UV light. The washed cell pellets were solubilized in 150 mmol/L NaCl, 100 mmol/L KCl, 2 mmol/L EDTA, and 10 mmol/L Hepes pH 7.4 containing 1% N-octylglucoside and the protease inhibitors DFP, PMSF, aprotinin, bestatin, chymostatin, and pepstatin. Cell lysates were then incubated with avidin-Sepharose for 2 hours followed by washing and denaturation of the avidin beads with reduced sodium dodecyl sulfate (SDS) sample buffer. Samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) on 6% to 16% polyacrylamide gradient gels followed by western blot, probed with peroxidase-conjugated streptavidin, and developed by enhanced chemiluminescence (Amersham Inc). Each lane represents the biotin-labeled protein profiles of ∼3 × 106 cells. Molecular weights in kiloDaltons are shown to the side. As can be seen, specific labeling of PMN protein bands in the 30 to 60 kD range was inhibited by coincubation with unlabeled peptide, whereas no specific labeling is observed for HT29 cells.
