3.10 Partitioning
of Hydrophobic
and Hydrophilic
Proteins Using
Triton X-114
1. Resuspend protein sample in 4% TX-114 (see Note 70) and
transfer to an Oakridge centrifuge tube. If starting from a
sample that is already suspended in PBS, add 0.125 mL of
32% TX-114 stock solution per 1 mL of PBS/protein solution
to bring the total solution to 4%. Rock at 4
C for 8–16 h.
2. Centrifuge at 27,000 Â g, 4
C for 1 h. Collect the supernatant
into clean centrifuge tubes and place at 4
C for later use.
3. Resuspend the pellets in 4% TX-114 and incubate with rocking
at 4
C for 1–2 h; repeat step 2.
4. Combine the two supernatants and repeat centrifugation to
remove any remaining insoluble material. Transfer the supernatant to new centrifuge tubes(s) and repeat centrifugation
until no visible pellet is obtained.
5. Incubate tubes containing the final clarified supernatant in a
37
C water bath until a partition appears (1–2 h).
6. Centrifuge at 27,000 Â g, at 25–37
C for 1 h (see Note 71).
7. Using a pipette, remove the upper aqueous layer, making note
of the volume being removed. Be sure to remove all of the
aqueous material, even if there is some loss of the lower
TX-114 layer.
8. To the TX-114 layer, add a volume of PBS equal to that
removed.
9. Repeat steps 5–8.
10. Repeat steps 5–7.
11. To the final TX-114 layer, add approximately 9 volumes of
ice-cold acetone per 1 volume of TX-114 layer, and place the
samples at À20
C for 8–16 h.
12. Centrifuge the samples at 27,000 Â g, 4
C for 1 h. Decant the
supernatant and dispose as hazardous waste.
13. Wash the pellet with ice-cold acetone, repeat centrifugation,
decant, and dispose supernatant as hazardous waste.
14. Remove residual acetone by applying a gentle stream of nitrogen using a nitrogen bath, or by leaving the tubes open in a
chemical fume hood until dry.
15. Resuspend each acetone precipitate in 30 mL of PBS. It may be
necessary to gently scrape the pellet from the side of the
centrifuge tube and to slowly stir on a stir plate. The pellet
will not fully go into solution.
16. Split each 30 mL sample between two 50 mL Oakridge centrifuge tubes. Use a glass pipette to add 15 mL of PBS-saturated
phenol to each sample in a chemical fume hood (see Note 72).
Rock at RT for 4 h.
17. Centrifuge at 27,000 Â g, 25
C for 1 h.
Extraction and Separation of Mycobacterial Proteins
97
of Hydrophobic
and Hydrophilic
Proteins Using
Triton X-114
1. Resuspend protein sample in 4% TX-114 (see Note 70) and
transfer to an Oakridge centrifuge tube. If starting from a
sample that is already suspended in PBS, add 0.125 mL of
32% TX-114 stock solution per 1 mL of PBS/protein solution
to bring the total solution to 4%. Rock at 4
C for 8–16 h.
2. Centrifuge at 27,000 Â g, 4
C for 1 h. Collect the supernatant
into clean centrifuge tubes and place at 4
C for later use.
3. Resuspend the pellets in 4% TX-114 and incubate with rocking
at 4
C for 1–2 h; repeat step 2.
4. Combine the two supernatants and repeat centrifugation to
remove any remaining insoluble material. Transfer the supernatant to new centrifuge tubes(s) and repeat centrifugation
until no visible pellet is obtained.
5. Incubate tubes containing the final clarified supernatant in a
37
C water bath until a partition appears (1–2 h).
6. Centrifuge at 27,000 Â g, at 25–37
C for 1 h (see Note 71).
7. Using a pipette, remove the upper aqueous layer, making note
of the volume being removed. Be sure to remove all of the
aqueous material, even if there is some loss of the lower
TX-114 layer.
8. To the TX-114 layer, add a volume of PBS equal to that
removed.
9. Repeat steps 5–8.
10. Repeat steps 5–7.
11. To the final TX-114 layer, add approximately 9 volumes of
ice-cold acetone per 1 volume of TX-114 layer, and place the
samples at À20
C for 8–16 h.
12. Centrifuge the samples at 27,000 Â g, 4
C for 1 h. Decant the
supernatant and dispose as hazardous waste.
13. Wash the pellet with ice-cold acetone, repeat centrifugation,
decant, and dispose supernatant as hazardous waste.
14. Remove residual acetone by applying a gentle stream of nitrogen using a nitrogen bath, or by leaving the tubes open in a
chemical fume hood until dry.
15. Resuspend each acetone precipitate in 30 mL of PBS. It may be
necessary to gently scrape the pellet from the side of the
centrifuge tube and to slowly stir on a stir plate. The pellet
will not fully go into solution.
16. Split each 30 mL sample between two 50 mL Oakridge centrifuge tubes. Use a glass pipette to add 15 mL of PBS-saturated
phenol to each sample in a chemical fume hood (see Note 72).
Rock at RT for 4 h.
17. Centrifuge at 27,000 Â g, 25
C for 1 h.
Extraction and Separation of Mycobacterial Proteins
97