Samples of extracellular proteins (23 μg) were separated on 10% NuPAGE gels (Invitrogen, USA) by lithium dodecyl sulfate (LDS) polyacrylamide gel electrophoresis (PAGE). Upon electrophoresis, the separated proteins were stained with SimplyBlue SafeStain (Life Technologies, CA).
For Western blotting analyses of LtxA production, the separated proteins were transferred from the gel to a Protran nitrocellulose transfer membrane (Whatman, Germany) by semidry blotting. Subsequently, the membrane was incubated with the LtxA-specific monoclonal antibody 83 (30 (link)). LtxA-specific protein bands were visualized with an IRDye 800CW-labeled secondary goat-anti mouse antibody and scanning of the membrane with an Amersham Typhoon biomolecular imager (Cytiva, Medemblik, the Netherlands).
For Western blotting analyses of LtxA production, the separated proteins were transferred from the gel to a Protran nitrocellulose transfer membrane (Whatman, Germany) by semidry blotting. Subsequently, the membrane was incubated with the LtxA-specific monoclonal antibody 83 (30 (link)). LtxA-specific protein bands were visualized with an IRDye 800CW-labeled secondary goat-anti mouse antibody and scanning of the membrane with an Amersham Typhoon biomolecular imager (Cytiva, Medemblik, the Netherlands).
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