For phage ELISA, wells of Costar EIA RIA high binding plates were coated with antigen in 100 mM selleck chemical NaHCO3 pH 8. 5 at room temperature for 1 hr or at 4 C overnight. The well solu tions were decanted and unbound sites were blocked Inhibitors,Modulators,Libraries by incubation with PBS containing 1% BSA for 1 hr. The wells were washed with PBS containing 0. 05% Tween 20, then the phage solutions were added and allowed to bind at room temperature for 0. 5 1 hr. The phage so lutions were decanted, the wells washed 5 7 times with PBS T, then a solution containing anti M13 horseradish peroxidase conjugate was added and allowed to bind for 0. 5 1 hr as directed by the manufacturer. The wells were washed with PBS T and developed by addition of a 3, 3, 5, 5 Tetramethylbenzidine substrate.
The ELISA signal was quantified either by direct measurement of blue color absorbance or by quenching with H2SO4 after 10 mins and Inhibitors,Modulators,Libraries determin ing the OD at 450 nm. Library construction Library DNA was prepared using Kunkel mutagenesis. A template clone based on pJH3B was prepared in which LCDR2 and LCDR3 regions were replaced with poly rare Arg codon containing segments. We have found that rare Arg codon containing segments provide enhanced selection relative to similar strategies that use stop codon containing template clones because the residual rare Arg codon template is less prone to growth advantages. Single stranded, uridine enriched DNA of rare Arg containing template clone was prepared in CJ2036 E. coli using established protocols. Kunkel mutagenesis performed using 5 phosphorylated primers corresponding to the reverse complement of the designed library sequences as previ ously described.
In general, Kunkel reactions contained 10 ug of template DNA, three fold excess of library pri mer, three units of T7 polymerase and two units of T4 lig ase. These reactions were incubated at room temperature overnight and then the library DNA purified using a QIAgen PCR purification kit. The E. coli Anacetrapib clone SS320 was used for library electroporations and was prepared by mating MC1016 and XL1 Blue. The purified library DNA was electroporated into SS320 competent cells that had been preinfected with VCSM13 or K07. Typical electroporations were performed with 350 Inhibitors,Modulators,Libraries uL of competent cells and 10 ug of purified library DNA in 0. 2 cm cuvettes using a BioRad Gene Pulser electroporator.
Cells were allowed to recover for 45 min at 37 C and Inhibitors,Modulators,Libraries then large scale phage production was performed as above. Library phage were suspended in PBS and either used immediately for screening or stored at 80 C. The final library phage pre parations had high infectious titer. The quality was assessed by large scale DNA sequencing of phage clones, in all cases, the libraries were highly di verse in sequence and contained 30% functional library members. Library selection and analysis Library sorting was performed in Costar EIA RIA plates, the antigen was immobilized http://www.selleckchem.com/products/arq-197.html into plate wells as above.
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