23. Remove the samples from their dialysis membranes and transfer to clean sample tubes.
24. Quantitate the amount of protein by BCA assay (see Note 11).
Based on the assay results, run 3–4μg of each product on
SDS-PAGE gel to visualize protein bands.
25. Store the sample at À80
C.
3.7 Extraction
of Proteins from
Subcellular Fractions
Using SDS [19]
1. Heat 600 mL of pyrogen-free, 0.2μm filtered water to 60
C
on a heated stir plate.
2. Prepare protein sample for extraction (for whole-cell lysate see
Subheading 3.4 or 3.5, for cell wall or membrane see Subheading 3.6) by transferring sample into a sterile 50 mL Oakridge
centrifuge tube; remove as much aqueous solution as possible
(by drying or centrifugation).
3. Add 30 mL of 2% SDS in PBS (w/v).
4. Add micro stir bars to samples.
5. Cap tubes and place in heated water from step 1 for 1 h,
ensuring the sample is stirring throughout extraction
procedure.
6. Remove Oakridge tubes from heated water bath and let cool
to RT.
7. Remove stir bars.
8. Centrifuge the sample at 27,000 Â g, 22
C for 30 min.
9. Collect the supernatant and reserve at 4
C.
10. Repeat steps 3–9 three additional times. SDS-soluble protein
is now extracted. Continue to steps 11–17 if removal of SDS is
required for downstream applications.
11. Pool the reserved supernatants into a large glass cylinder or
beaker sufficient to permit dilution of sample 20-fold; this is
necessary to effectively remove SDS detergent by paired-ion
extraction [20].
12. To perform a paired-ion extraction, add 17 mL of acetone,
10 mL of glacial acetic acid, and 10 mL of triethylamine for
every 10 mL of pooled SDS-soluble protein extract and incubate at À20
C for 4–16 h.
13. Decant organic solvent (discard as hazardous waste) until a
concentrated slurry can be recovered and transferred to a
50 mL Oakridge tube.
14. Centrifuge the protein slurry at 27,000 Â g, 22
C for 30 min.
15. Decant organic solvent (dispose as hazardous waste); add
30 mL of pyrogen-free, 0.2μm filtered water to pellet and
wash gently.
16. Centrifuge the protein solution at 27,000 Â g, 22
C for
30 min.
Extraction and Separation of Mycobacterial Proteins
95
24. Quantitate the amount of protein by BCA assay (see Note 11).
Based on the assay results, run 3–4μg of each product on
SDS-PAGE gel to visualize protein bands.
25. Store the sample at À80
C.
3.7 Extraction
of Proteins from
Subcellular Fractions
Using SDS [19]
1. Heat 600 mL of pyrogen-free, 0.2μm filtered water to 60
C
on a heated stir plate.
2. Prepare protein sample for extraction (for whole-cell lysate see
Subheading 3.4 or 3.5, for cell wall or membrane see Subheading 3.6) by transferring sample into a sterile 50 mL Oakridge
centrifuge tube; remove as much aqueous solution as possible
(by drying or centrifugation).
3. Add 30 mL of 2% SDS in PBS (w/v).
4. Add micro stir bars to samples.
5. Cap tubes and place in heated water from step 1 for 1 h,
ensuring the sample is stirring throughout extraction
procedure.
6. Remove Oakridge tubes from heated water bath and let cool
to RT.
7. Remove stir bars.
8. Centrifuge the sample at 27,000 Â g, 22
C for 30 min.
9. Collect the supernatant and reserve at 4
C.
10. Repeat steps 3–9 three additional times. SDS-soluble protein
is now extracted. Continue to steps 11–17 if removal of SDS is
required for downstream applications.
11. Pool the reserved supernatants into a large glass cylinder or
beaker sufficient to permit dilution of sample 20-fold; this is
necessary to effectively remove SDS detergent by paired-ion
extraction [20].
12. To perform a paired-ion extraction, add 17 mL of acetone,
10 mL of glacial acetic acid, and 10 mL of triethylamine for
every 10 mL of pooled SDS-soluble protein extract and incubate at À20
C for 4–16 h.
13. Decant organic solvent (discard as hazardous waste) until a
concentrated slurry can be recovered and transferred to a
50 mL Oakridge tube.
14. Centrifuge the protein slurry at 27,000 Â g, 22
C for 30 min.
15. Decant organic solvent (dispose as hazardous waste); add
30 mL of pyrogen-free, 0.2μm filtered water to pellet and
wash gently.
16. Centrifuge the protein solution at 27,000 Â g, 22
C for
30 min.
Extraction and Separation of Mycobacterial Proteins
95