title: OspC Protein Microarray v2
status: Public on Aug 22 2013
contact.city: Irvine
contact.first: Elisabeth
contact.last: Baum
contact.state: CA
contact.address: Med Surge II
contact.country: USA
contact.institute: University of California Irvine
organism: Borreliella burgdorferi
manufacture_protocol: Primers used for amplifying full-length ospC alleles had sequences corresponding to positions 16903 through 16925 (Forward) and 17529 through 17558 (Reverse) of cp26 of the B31 strain. For amplification of subcloning targets for protein expression the Forward primer eliminated the signal peptide and 4 different Reverse primers  (to accommodate 3’ end nucleotide polymorphisms amongst ospC alleles), corresponding to nucleotide positions 614 through 633 were used. Sequence-verified clones of full-length ospC alleles were subcloned into a pXT7 expression vector containing an amino-terminal 10X-Histidine (His) fusion tag. Plasmids were transformed into E. coli BL21(DE3)pLysS (Promega, Madison, WI) for protein expression in Terrific Broth (MP Biomedicals, Solon, OH) induced with IPTG (RPI, Mt. Prospect, IL), lysed and  centrifuged. Cleared bacterial lysate was incubated with Ni-coupled magnetic beads (MagneHis kit, Promega, Madison, WI). Purified recombinant proteins were eluted from beads and sample purity was estimated by densitometry of coomassie-blue stained protein bands on sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels; protein concentration was determined by BCA Protein Assay kit (Pierce, Rockford, IL). Purified recombinant-OspC proteins were printed on nitrocellulose-coated glass FAST slides (Whatman, Piscataway, NJ) using an Omnigrid 100 apparatus (Digilab, Holliston, MA), in duplicate spots and 2 different amounts of approximately 10 pg and 30 pg of protein per spot. Protein storage buffer alone was printed on the array to serve as a background signal control.