2.10 Partitioning
of Hydrophobic
and Hydrophilic
Proteins Using
Triton X-114
1. Soluble protein sample (such as CFP obtained in Subheading
3.2. or whole-cell lysate obtained in Subheading 3.4 or 3.5).
2. Phosphate-buffered saline (PBS) (see Subheading 2.3, item 4).
3. 32% TX-114 stock solution: Combine equal parts of Triton
X-114 and PBS. Stir at RT for several hours. Transfer to 4
C
and let stand until the mixture clears, which may take overnight
(if it does not clear, it was not mixed thoroughly enough).
Transfer the mixture to a 37
C water bath or incubator until
a biphase appears (see Note 21). Carefully remove and discard
the top layer being sure not to disrupt the bottom Triton layer.
Take note of the volume of Triton and add an equal volume of
PBS. Stir at RT until the mixture clears, then incubate at 37
C
until a biphase appears. Remove the PBS layer and double the
volume again. Repeat the stirring and incubation and remove
the PBS layer. Stir the final Triton layer on a stir plate to
recombine any PBS not removed. The final Triton layer is the
32% TX-114.
4. 4% TX-114 in PBS: Dilute 12.5 mL 32% TX-114 stock solution to 100 mL total in PBS.
5. Polypropylene centrifuge bottles.
6. Platform mixer.
7. High-speed centrifuge and rotors.
8. 37
C water bath.
9. Acetone, stored at À20
C (see Note 22).
10. Nitrogen bath.
11. Oakridge centrifuge tubes (Teflon), 50 mL, sterile.
12. PBS-saturated phenol (see Note 23): Working in a chemical
fume hood, resuspend phenol crystals in PBS at 50%
w/v. Incubate in 55
C water bath until crystals melt and a
partition forms. Carefully remove the top PBS layer using a
glass pipet and discard as hazardous waste. Add an equal volume of PBS back to the phenol layer. Repeat incubation and
removal of top layer two more times. Keep the bottom layer as
PBS-saturated phenol.
13. Dialysis tubing, 3.5 kDa MWCO.
14. Dialysis clips and foam float.
15. Dialysis tank (7 L).
16. Pyrogen-free, 0.2μm filtered water.
17. SDS-PAGE gel.
18. Bicinchoninic acid (BCA) protein assay kit (see Note 11).
Extraction and Separation of Mycobacterial Proteins
83
of Hydrophobic
and Hydrophilic
Proteins Using
Triton X-114
1. Soluble protein sample (such as CFP obtained in Subheading
3.2. or whole-cell lysate obtained in Subheading 3.4 or 3.5).
2. Phosphate-buffered saline (PBS) (see Subheading 2.3, item 4).
3. 32% TX-114 stock solution: Combine equal parts of Triton
X-114 and PBS. Stir at RT for several hours. Transfer to 4
C
and let stand until the mixture clears, which may take overnight
(if it does not clear, it was not mixed thoroughly enough).
Transfer the mixture to a 37
C water bath or incubator until
a biphase appears (see Note 21). Carefully remove and discard
the top layer being sure not to disrupt the bottom Triton layer.
Take note of the volume of Triton and add an equal volume of
PBS. Stir at RT until the mixture clears, then incubate at 37
C
until a biphase appears. Remove the PBS layer and double the
volume again. Repeat the stirring and incubation and remove
the PBS layer. Stir the final Triton layer on a stir plate to
recombine any PBS not removed. The final Triton layer is the
32% TX-114.
4. 4% TX-114 in PBS: Dilute 12.5 mL 32% TX-114 stock solution to 100 mL total in PBS.
5. Polypropylene centrifuge bottles.
6. Platform mixer.
7. High-speed centrifuge and rotors.
8. 37
C water bath.
9. Acetone, stored at À20
C (see Note 22).
10. Nitrogen bath.
11. Oakridge centrifuge tubes (Teflon), 50 mL, sterile.
12. PBS-saturated phenol (see Note 23): Working in a chemical
fume hood, resuspend phenol crystals in PBS at 50%
w/v. Incubate in 55
C water bath until crystals melt and a
partition forms. Carefully remove the top PBS layer using a
glass pipet and discard as hazardous waste. Add an equal volume of PBS back to the phenol layer. Repeat incubation and
removal of top layer two more times. Keep the bottom layer as
PBS-saturated phenol.
13. Dialysis tubing, 3.5 kDa MWCO.
14. Dialysis clips and foam float.
15. Dialysis tank (7 L).
16. Pyrogen-free, 0.2μm filtered water.
17. SDS-PAGE gel.
18. Bicinchoninic acid (BCA) protein assay kit (see Note 11).
Extraction and Separation of Mycobacterial Proteins
83