EVALUATION OF DIFFERENT METHODS FOR THE DETECTION OF OUTER-MEMBRANE PROTEINS AND LIPOPOLYSACCHARIDES OF PASTEURELLA-HAEMOLYTICA BY IMMUNOBLOTTING

The optimal conditions for the detection of outer membrane proteins (O MPs) and lipopolysaccharide (LPS) of Pasteurella haemolytica by immuno blotting were evaluated. The variables examined included the equilibra tion time of the gels before transfer, composition of the transfer buf fer, type of blotting membrane, blocking agent, effect of the zwitteri onic detergent Empigen BB on protein renaturation, and the development reagent. The composition of the transfer buffer and time of gel equil ibration significantly affected the efficiency of transfer of both OMP s and LPS. However, the optimal conditions for the transfer of OMPs we re not the same as those for LPS. Thus, optimal transfer of OMPs occur red in Tris-glycine buffer, with prior equilibration of the gels to al low for expansion, whereas optimal transfer of LPS was achieved in Tri s-glycine-methanol buffer with no equilibration of the gels. In Tris-g lycine-methanol buffer, gel equilibration resulted in a significantly reduced transfer of both OMPs and LPS, probably due to the removal of SDS from these components. The use of Zeta-Probe blotting membrane whi ch, unlike nitrocellulose, does not require methanol for optimal prote in binding, did not result in improved binding of OMPs or LPS in the a bsence of methanol and, even after prolonged blocking (>2 h), gave hig her background staining than did nitrocellulose. Effective blocking of nitrocellulose was achieved with 3%(w/v) gelatin, 2.5%(w/v) skimmed m ilk or 0.3%(v/v) Tween 20, whereas increased background staining occur red with 1%(w/v) bovine serum albumin or 1% (w/v) ovalbumin. The incor poration of Empigen BB in the primary antibody buffer did not improve antibody recognition of proteins as a result of their renaturation. Fo r the horseradish-peroxidase enzyme development system, the substrate 3,3'-diaminobenzidine tetrahydrochloride was more sensitive, and devel oped more quickly, than 4-chloro-1-naphthol, but faded more rapidly af ter drying of the membrane. 4-chloro-1-naphthol was more suitable for identifying OMPs because less background staining occurred, whereas 3, 3'-diaminobenzidine tetrahydrochloride was more suitable for the detec tion of LPS due to its greater sensitivity.