Mycobacterium avium subsp. paratuberculosis (Map), an extremely hardy organism, due mainly to the chemical composition of its thick, waxy outer cell wall has been shown to survive thermal treatments such as pasteurisation. Investigation of this thermal resistance at a proteomic level may give an insight into the stress tolerance of this bacterium and may help develop better diagnostics for the detection of this organism for animal disease control. This study assessed the efficacy of a number of lysis/extraction techniques to release intracellular proteins from Map strains for proteomic analysis by 2 dimensional electrophoresis (2DE).Map cells were harvested, washed and lysed/disrupted using each of the following methods: Bugbuster (Novagen); Fast Protein Blue; Fast Protein Red (Qbiogene ); B-Per, Y-Per - (Perbio Sciences), all chemical treatments, and a sonication procedure including glass beads and CHAPS detergent. Map lysates were prepared for 2DE by dilution in isoelectric focusing (IEF) rehydration solution on pH 3-10 IPG strips and focused on the Multiphor 11 apparatus. Second dimension electrophoresis was performed on precast SDS- polyacrylamide gels. Silver stained proteins were analysed using Phoretix 2D software.Comparison of the methods using strain NCTC 8578 indicated that sonication with glass beads and CHAPS detergent yielded the greatest quantity of protein of all methods tested. Lysis of Map with Bugbuster was the least efficient of the methods employed.A sonication method incorporating glass beads and the detergent CHAPS was a more effective lysis protocol than a number of commercially available chemical-based methods for the release of the Map proteome. The effective extraction of the Map proteome, through the developed method, will assist the investigation of the thermal tolerance of this organism at the proteomic level. Also, the availability of a more complete proteome will be critical to developing better diagnostics and /or vaccines for disease detection, treatment and control.