59. Set speed to maximum (setting 48). Do not set time on Bead
Beater. Use timer to measure length of beater session.
60. To maintain sample integrity for downstream applications, it is
important to keep the samples cool at all times and perform
work quickly prior to loss of activity from protease inhibitor
cocktails.
61. Acid-fast staining can be performed at various stages to assess
progression of cellular lysis; prepare slides in a biosafety cabinet
if assessing the breakage of live cells.
62. After centrifugation, you will see the silica in the bottom of the
tube, followed by semi-cleared lysate, then a “foamy” top layer
of cellular debris.
63. This centrifugation step can be modified to pellet cell wall
material which makes the subsequent lysate more amenable
to filter sterilization. Filter sterilization will permit transfer of
material out of the BSL-3 for downstream use. A suggested
centrifugation is 25,000 Â g for 20 min. If this step is performed, the supernatant will contain cytosol and membrane,
and the pellet will contain cell wall material. The pelleted
material may be inactivated by gamma irradiation or heat-killed
for use outside of the BSL-3.
64. If using lysate generated from Subheading 3.4, double the
French press lysate volume.
65. Supernatant is whole-cell lysate (WCL). If WCL is one of the
desired products, remove desired quantity, dialyze, and store.
Use remaining supernatant to continue with protocol.
66. Centrifuge tubes must be filled to manufacturer’s specified
volume in order to prevent collapse, and they must be balanced
to within 100 mg using an electronic balance.
67. Wash the pellet before storing. Waiting to wash the pellet makes
it more likely that the pellet will be disrupted during washing.
If the pellets are disrupted during the washing step, fill the
centrifuge tubes with breaking buffer, balance, and centrifuge
as in steps 10 and 11.
68. Dialysis tubes should be identified with its contents by labeling
the clip or float, so final products are not confused if multiple
samples are in one tank.
69. The step-dilution dialysis for urea helps prevent the urea from
precipitating out of solution.
70. If partitioning whole-cell lysate, TX-114 may be added to the
breaking buffer (see Subheading 2.4, item 6) to a final concentration of 4%; this procedure can be initiated at the conclusion
of Subheading 3.4.
Extraction and Separation of Mycobacterial Proteins
105
Beater. Use timer to measure length of beater session.
60. To maintain sample integrity for downstream applications, it is
important to keep the samples cool at all times and perform
work quickly prior to loss of activity from protease inhibitor
cocktails.
61. Acid-fast staining can be performed at various stages to assess
progression of cellular lysis; prepare slides in a biosafety cabinet
if assessing the breakage of live cells.
62. After centrifugation, you will see the silica in the bottom of the
tube, followed by semi-cleared lysate, then a “foamy” top layer
of cellular debris.
63. This centrifugation step can be modified to pellet cell wall
material which makes the subsequent lysate more amenable
to filter sterilization. Filter sterilization will permit transfer of
material out of the BSL-3 for downstream use. A suggested
centrifugation is 25,000 Â g for 20 min. If this step is performed, the supernatant will contain cytosol and membrane,
and the pellet will contain cell wall material. The pelleted
material may be inactivated by gamma irradiation or heat-killed
for use outside of the BSL-3.
64. If using lysate generated from Subheading 3.4, double the
French press lysate volume.
65. Supernatant is whole-cell lysate (WCL). If WCL is one of the
desired products, remove desired quantity, dialyze, and store.
Use remaining supernatant to continue with protocol.
66. Centrifuge tubes must be filled to manufacturer’s specified
volume in order to prevent collapse, and they must be balanced
to within 100 mg using an electronic balance.
67. Wash the pellet before storing. Waiting to wash the pellet makes
it more likely that the pellet will be disrupted during washing.
If the pellets are disrupted during the washing step, fill the
centrifuge tubes with breaking buffer, balance, and centrifuge
as in steps 10 and 11.
68. Dialysis tubes should be identified with its contents by labeling
the clip or float, so final products are not confused if multiple
samples are in one tank.
69. The step-dilution dialysis for urea helps prevent the urea from
precipitating out of solution.
70. If partitioning whole-cell lysate, TX-114 may be added to the
breaking buffer (see Subheading 2.4, item 6) to a final concentration of 4%; this procedure can be initiated at the conclusion
of Subheading 3.4.
Extraction and Separation of Mycobacterial Proteins
105