Method for identification of proteins from intracellular bacteria

A method for identifying proteins secreted from an intracellular bacteria, comprising the following steps:

• 1) infecting host cells by the intracellular bacteria,
• 2) pulse labelling of the intracellular bacteria present in the infected cells,
• 3) preparing whole cell lysates of the infected cells after different periods of chase following step 2),
• 4) comparing 2D-gel electrophoresis protein profiles of the whole cell lysates prepared after different periods of chase from step 3),
• 5) detecting protein spots from step 4) which are present in decreasing amount as chasing periods increase in step 3),
• 6) identifying the proteins in the spots selected in step 5).

A method for identifying proteins secreted from an intracellular bacteria, comprising the following steps:

• 1) infecting host cells by the intracellular bacteria,
• 2) cultivating the host cells in the presence and in the absence of a proteasome inhibitor, respectively,
• 3) labelling of the intracellular bacteria present in the infected cells cultivated in the presence and in the absence of a proteasome inhibitor, respectively,
• 4) preparing whole cell lysates of the infected cells,
• 5) comparing 2D-gel electrophoresis protein profiles of the whole cell lysates of the infected cells cultivated in the presence and in the absence of a proteasome inhibitor, respectively,
• 6) detecting protein spots from step 5) which are present in the whole cell lysates cultivated in the presence of a proteasome inhibitor, but absent or present in significantly reduced amount in the whole cell lysates cultivated in the absence of a proteasome inhibitor,
• 7) identifying the proteins in the spots selected in step 6). decreasing amount as chasing periods increase in step 3),
• 8) identifying the proteins in the spots selected in step 5).