38. Ensure the pressure on the nitrogen regulator does not exceed
limits specified by ultrafiltration membrane and stirred
ultrafiltration cell.
39. If a leak is found or the output flow is too great, shut off the
flow of nitrogen and release pressure from the stainless-steel
dispensing pressure vessel, then from the stirred cell. Leaks at
the lid or base are generally caused by poorly seated O-rings.
Leaks from tubing connections may require tightening, or the
use of Teflon tape. If the output flow is too fast, this could
indicate a poorly seated ultrafiltration membrane or O-ring, or
a flaw in the membrane. Once the problem is corrected, repeat
startup procedures.
40. To ensure complete exchange of the buffer, 20 times the CFP
volume of 10 mM ammonium bicarbonate should be added to
the reservoir. For example, if there is 100 mL of CFP remaining, 2 L of 10 mM ammonium bicarbonate should be added to
the stainless-steel dispensing pressure vessel.
41. Suggested antibodies for M. tuberculosis CFP analysis by Western blot include anti-GroES (Rv3418c), anti-SodA (Rv3846),
anti-Ag85 complex (Rv0129c, Rv1886c, and Rv3804c), antiPstS1 (Rv0934), and anti-DnaK (Rv0350) as positive controls
and anti-GroEL2 (Rv0440) as a negative control. These and
other anti-Mycobacterium antibodies are available through an
NIH/NIAID biological reagent repository such as BEI
Resources (http://www.beiresources.org/).
42. The starting product can be transitioned directly upon completion of the Culture filtrate protocol (see Subheading 3.2)
prior to freezing, eliminating the need for thawing.
43. The eluate is considered vesicle-depleted CFP, which may be of
interest when evaluating the quality of vesicle enrichment.
44. The maximum protein-binding capacity for Capto™ Core
700 is 14 mg/mL of resin.
45. Suggested antibodies for M. tuberculosis MEV analysis by Western blot include anti-GlcB (Rv1837c), anti-LAM (Rv), antiLpqH (Rv3763) as positive controls, and anti-GroES
(Rv3418c) as a negative control. These and other anti-Mycobacterium antibodies are available through an NIH/NIAID
biological reagent repository such as BEI Resources (http://
www.beiresources.org/).
46. If the sample is too concentrated for accurate analysis, perform
1:10 serial dilutions from the 5μg sample in 1 mL PBS until an
appropriate concentration is achieved. In contrast, if the sample
is too dilute, an additional 100 kDa concentration may be
performed (see Subheading 3.3, steps 2–6), followed by microBCA assay.
Extraction and Separation of Mycobacterial Proteins
103
limits specified by ultrafiltration membrane and stirred
ultrafiltration cell.
39. If a leak is found or the output flow is too great, shut off the
flow of nitrogen and release pressure from the stainless-steel
dispensing pressure vessel, then from the stirred cell. Leaks at
the lid or base are generally caused by poorly seated O-rings.
Leaks from tubing connections may require tightening, or the
use of Teflon tape. If the output flow is too fast, this could
indicate a poorly seated ultrafiltration membrane or O-ring, or
a flaw in the membrane. Once the problem is corrected, repeat
startup procedures.
40. To ensure complete exchange of the buffer, 20 times the CFP
volume of 10 mM ammonium bicarbonate should be added to
the reservoir. For example, if there is 100 mL of CFP remaining, 2 L of 10 mM ammonium bicarbonate should be added to
the stainless-steel dispensing pressure vessel.
41. Suggested antibodies for M. tuberculosis CFP analysis by Western blot include anti-GroES (Rv3418c), anti-SodA (Rv3846),
anti-Ag85 complex (Rv0129c, Rv1886c, and Rv3804c), antiPstS1 (Rv0934), and anti-DnaK (Rv0350) as positive controls
and anti-GroEL2 (Rv0440) as a negative control. These and
other anti-Mycobacterium antibodies are available through an
NIH/NIAID biological reagent repository such as BEI
Resources (http://www.beiresources.org/).
42. The starting product can be transitioned directly upon completion of the Culture filtrate protocol (see Subheading 3.2)
prior to freezing, eliminating the need for thawing.
43. The eluate is considered vesicle-depleted CFP, which may be of
interest when evaluating the quality of vesicle enrichment.
44. The maximum protein-binding capacity for Capto™ Core
700 is 14 mg/mL of resin.
45. Suggested antibodies for M. tuberculosis MEV analysis by Western blot include anti-GlcB (Rv1837c), anti-LAM (Rv), antiLpqH (Rv3763) as positive controls, and anti-GroES
(Rv3418c) as a negative control. These and other anti-Mycobacterium antibodies are available through an NIH/NIAID
biological reagent repository such as BEI Resources (http://
www.beiresources.org/).
46. If the sample is too concentrated for accurate analysis, perform
1:10 serial dilutions from the 5μg sample in 1 mL PBS until an
appropriate concentration is achieved. In contrast, if the sample
is too dilute, an additional 100 kDa concentration may be
performed (see Subheading 3.3, steps 2–6), followed by microBCA assay.
Extraction and Separation of Mycobacterial Proteins
103