). The bacterial debris was pelleted by centrifugation at 16,000 rpm

for 30 minutes, and the soluble fraction was applied to Ni-NTA agarose resin (Qiagen Inc.). After incubation at 4°C for 30–60 minutes, the resin was spun down at 1000xg for 60s. The pelleted resin was added to an empty column and washed by gravity flow with copious amounts of lysis buffer. Protein was eluted off the Ni-NTA resin in a buffer containing 20 mM HEPES pH 7.3, 150 mM NaCl, and 300 mM Imidazole. Further purification was performed by Size Crizotinib Exclusion Chromatography (SEC) using a 320 ml Sephadex 200 column (GE lifesciences) in a buffer consisting of 20 mM HEPES 7.3, 150 mM NaCl, and 5% (v/v) glycerol. Fractions containing the scFv were pooled, aliquoted, flash frozen in liquid nitrogen, and stored at -80°C. Binding efficiency for flash frozen scFv versus unfrozen scFv were compared and the binding was identical (data not shown) demonstrating that the freezing the protein for long term storage did not alter binding capacity. Binding specificity assay Purified, recombinant scFv was used to test specificity for L. acidophilus. Before the assay, the scFv was incubated with an excess of GFP1-10 complementary protein as described previously [37] ON at 4°C. The following day 5–15 μg of scFv with or without restored GFP were incubated with 106-107 bacteria Akt inhibitor in solution

containing PBS and Wash Buffer (0.5% BSA, 2 mM EDTA). After 1 h incubation at RT the bacteria were washed twice with PBS and resuspended in a 1:1000–1:2000 anti-SV5-PE (1 μg/μl). Incubation was performed for 1 h at RT and the cells were washed and resuspended in PBS prior to analysis with two different flow cytometers. The BD LSRII was used to evaluate the mean average fluorescence for binding activity of the scFv, and the AMNIS was used to image fluorescently labeled scFv bound to cells. The same procedure was followed for the other Lactobacillus species

and for the other species selleck chemicals llc to clearly confirm the specificity of the scFv binding. Capture efficiency assay Individual bacteria species (Table 1) were grown separately, washed, and all diluted in PBS to an OD600 of 1.0 where an absorbance of 1.0 is equal to ~109 bacteria cells per milliliter. Equal volumes of each bacteria were mixed with L. acidophilus added at theoretical ratios of 10%, 5%, 1%, and 0.1%. α-La was prepared and incubated with bacterial mixtures as described above. Samples were analyzed on BD Influx. Three gates were used for the analysis: P1, P2, and P3. P1 was drawn to include bacteria defined by size and morphology using a two dimensional Side Scatter (SSC):Forward Scatter (FSC) plot. P2 and P3 are drawn in a two dimensional fluorescence (FITC:PE) plot and include bacteria captured in the P1 gate. P3 is drawn using a control sample consisting solely of L.