2.11 Partitioning
of Hydrophobic
and Hydrophilic
Proteins Using
Ammonium Sulfate
1. Soluble protein sample (such as CFP obtained in Subheading
3.2).
2. Ammonium sulfate, (NH 4 ) 2 SO 4 .
3. Dialysis tubing, 3.5 kDa MWCO.
4. Dialysis tank (7 L).
5. Dialysis buffer #1: 10 mM NH 4 HCO 3 (see Subheading 2.2,
item 4), 1 mM DTT.
6. Dialysis buffer #2: 10 mM NH 4 HCO 3 (see Subheading 2.2,
item 4).
7. SDS-PAGE gel.
8. Bicinchoninic acid (BCA) protein assay kit (see Note 11).
9. Polypropylene centrifuge bottles.
10. High-speed centrifuge and rotor.
11. Freeze-dryer and flask.
3 Methods
3.1 M. tuberculosis
Inactivation by Gamma
Irradiation
M. tuberculosis biosafety consideration: M. tuberculosis is a human
risk-group 3 pathogen and requires that work with live organisms is
conducted in a BSL-3 laboratory, in accordance with the criteria for
a BSL-3 described in the Biosafety in Microbiology and Biomedical
Laboratories resource (https://www.cdc.gov/labs/BMBL.html).
Work in a BSL-3 laboratory requires specific training and approval
of a biosafety committee, biosafety officer, or similar governance
prior to commencement, based on country, territory, and institutional polices.
1. Remove the frozen M. tuberculosis live cell pellet from the
containment laboratory following institutional biosafety
approved practices.
2. Triple package vials or tubes containing the live cell pellet
inside biohazard bags with 2.5% Vesphene or suitable disinfectant in between each layer.
3. Wipe down the package containing the live cell pellet with 2.5%
Vesphene followed by 70% ethanol, before being placed in a
transfer area (see Note 24).
4. Transport package to designated radiation facility (see Note
25).
5. Using a JL Shepherd Model 81-14 sealed source Cs-137 irradiator, apply an irradiating dose to the pellet equivalent to 2.4
megarads (see Note 26).
6. The pellet should be transported back into the BSL-3 laboratory and must still be treated and transported as dangerous
goods (see Notes 24 and 27).
84
Megan Lucas et al.
of Hydrophobic
and Hydrophilic
Proteins Using
Ammonium Sulfate
1. Soluble protein sample (such as CFP obtained in Subheading
3.2).
2. Ammonium sulfate, (NH 4 ) 2 SO 4 .
3. Dialysis tubing, 3.5 kDa MWCO.
4. Dialysis tank (7 L).
5. Dialysis buffer #1: 10 mM NH 4 HCO 3 (see Subheading 2.2,
item 4), 1 mM DTT.
6. Dialysis buffer #2: 10 mM NH 4 HCO 3 (see Subheading 2.2,
item 4).
7. SDS-PAGE gel.
8. Bicinchoninic acid (BCA) protein assay kit (see Note 11).
9. Polypropylene centrifuge bottles.
10. High-speed centrifuge and rotor.
11. Freeze-dryer and flask.
3 Methods
3.1 M. tuberculosis
Inactivation by Gamma
Irradiation
M. tuberculosis biosafety consideration: M. tuberculosis is a human
risk-group 3 pathogen and requires that work with live organisms is
conducted in a BSL-3 laboratory, in accordance with the criteria for
a BSL-3 described in the Biosafety in Microbiology and Biomedical
Laboratories resource (https://www.cdc.gov/labs/BMBL.html).
Work in a BSL-3 laboratory requires specific training and approval
of a biosafety committee, biosafety officer, or similar governance
prior to commencement, based on country, territory, and institutional polices.
1. Remove the frozen M. tuberculosis live cell pellet from the
containment laboratory following institutional biosafety
approved practices.
2. Triple package vials or tubes containing the live cell pellet
inside biohazard bags with 2.5% Vesphene or suitable disinfectant in between each layer.
3. Wipe down the package containing the live cell pellet with 2.5%
Vesphene followed by 70% ethanol, before being placed in a
transfer area (see Note 24).
4. Transport package to designated radiation facility (see Note
25).
5. Using a JL Shepherd Model 81-14 sealed source Cs-137 irradiator, apply an irradiating dose to the pellet equivalent to 2.4
megarads (see Note 26).
6. The pellet should be transported back into the BSL-3 laboratory and must still be treated and transported as dangerous
goods (see Notes 24 and 27).
84
Megan Lucas et al.