Figure 2

Purification and Characterization of N-terminal fragment of P116 protein. A: SDS-PAGE analysis showing purification of N-terminal P116 (P116_(N-27)) protein on Ni-NTA column. Lane 1: Prestained standard protein marker, Lane 2: Protein extracts of uninduced E. coli, Lane 3: Protein extracts of induced (~27 KDa) E. coli, Lane 4: Flow through, Lanes 5&6: Wash1 & Wash2, Lane 7: Purified P116 protein before dialysis (eluted with buffer containing 8M Urea), Lane 8: Purified P116 protein after dialysis against buffer containing 0.5M Urea. B: Immunoblot analysis of purified P116_(N-27) protein with-(i) Healthy control human serum-Lane 1: Prestained standard protein marker, Lane 2: Protein extracts of uninduced E. coli, Lane 3: Recombinant protein detected with representative healthy human serum. (ii) M. pneumoniae antibodies-Lane 1: Recombinant protein detected using rabbit anti-M. pneumoniae serum Lane 2: Protein extracts of uninduced E. coli, Lane 3: Prestained standard protein marker. (iii) M. pneumoniae infected patient sera-Lanes 1&2: Recombinant protein detected using M. pneumoniae infected patient sera, Lane 3: Protein extracts of uninduced E. coli, Lane 4: Prestained standard protein marker. C: Immunoblot analysis of purified P116_(N-27) protein with patient sera infected with M. pneumoniae PM: Prestained standard protein marker, NC: Negative control (patient sample which tested negative with the reference test) and PC: positive control (patient sample which tested positive with the reference test), Lanes 1-11: patient sera infected with M. pneumoniae.