17. Repeat water wash and centrifugation (steps 15 and 16) two
additional times; remaining pellet, SDS-soluble protein fraction, may be used as needed for downstream applications.
3.8 Guanidine–HCl
Extraction
of Hydrophobic
Proteins
1. Prepare fresh 6 M Guanidine–HCl in pyrogen-free, 0.2μm
filtered water just prior to protein extraction (the resultant
solution will be colder than RT).
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). Add 30 mL of 6 M Guanidine–
HCl. Add micro stir bars to sample tubes. Cap tubes and place
on stir-plate.
3. Stir sample extractions at RT (22
C) for 1 h.
4. Remove stir bars. Centrifuge the protein extract at 27,000 Â g,
22
C for 30 min. Collect the supernatant into a new tube and
reserve at 4
C.
5. Add an additional 30 mL of 6 M Guanidine–HCl solution to
the remaining pellet, add stir bars, and repeat extraction and
centrifugation (steps 3 and 4) for a total of three extractions
per sample. Discard the final pellet.
6. Prepare dialysis tubing and exchange Guanidine–HCl extracts
pooled from step 5 into 10 mM ammonium bicarbonate (see
Subheading 3.6, steps 18–23 for instructions for dialysis).
3.9 8 M Urea
Extraction
of Hydrophobic
Proteins
1. 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). Add 30 mL of 8 M urea. Add
micro stir bars to sample tubes. Cap tubes and place on stirplate.
2. Stir sample extractions at RT (22
C) for 1 h.
3. Remove stir bars. Centrifuge the extracted protein at
27,000 Â g, 22
C for 30 min. Collect the supernatant into a
new tube and reserve at 4
C.
4. Add 30 mL of 8 M urea to the pellet, add stir bars, and repeat
extraction (steps 2 and 3) and centrifugation for a total of three
extractions per sample.
5. Prepare dialysis tubing (see Subheading 3.6, steps 18 and 19
for preparation of tubing for dialysis) and exchange 8 M urea
extracts first into 4 M urea at RT (22
C) for 4–16 h, followed
by exchange into 2 M urea at 4
C for 4–16 h. Perform final
dialysis into 10 mM ammonium bicarbonate (see Note 69).
96
Megan Lucas et al.
additional times; remaining pellet, SDS-soluble protein fraction, may be used as needed for downstream applications.
3.8 Guanidine–HCl
Extraction
of Hydrophobic
Proteins
1. Prepare fresh 6 M Guanidine–HCl in pyrogen-free, 0.2μm
filtered water just prior to protein extraction (the resultant
solution will be colder than RT).
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). Add 30 mL of 6 M Guanidine–
HCl. Add micro stir bars to sample tubes. Cap tubes and place
on stir-plate.
3. Stir sample extractions at RT (22
C) for 1 h.
4. Remove stir bars. Centrifuge the protein extract at 27,000 Â g,
22
C for 30 min. Collect the supernatant into a new tube and
reserve at 4
C.
5. Add an additional 30 mL of 6 M Guanidine–HCl solution to
the remaining pellet, add stir bars, and repeat extraction and
centrifugation (steps 3 and 4) for a total of three extractions
per sample. Discard the final pellet.
6. Prepare dialysis tubing and exchange Guanidine–HCl extracts
pooled from step 5 into 10 mM ammonium bicarbonate (see
Subheading 3.6, steps 18–23 for instructions for dialysis).
3.9 8 M Urea
Extraction
of Hydrophobic
Proteins
1. 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). Add 30 mL of 8 M urea. Add
micro stir bars to sample tubes. Cap tubes and place on stirplate.
2. Stir sample extractions at RT (22
C) for 1 h.
3. Remove stir bars. Centrifuge the extracted protein at
27,000 Â g, 22
C for 30 min. Collect the supernatant into a
new tube and reserve at 4
C.
4. Add 30 mL of 8 M urea to the pellet, add stir bars, and repeat
extraction (steps 2 and 3) and centrifugation for a total of three
extractions per sample.
5. Prepare dialysis tubing (see Subheading 3.6, steps 18 and 19
for preparation of tubing for dialysis) and exchange 8 M urea
extracts first into 4 M urea at RT (22
C) for 4–16 h, followed
by exchange into 2 M urea at 4
C for 4–16 h. Perform final
dialysis into 10 mM ammonium bicarbonate (see Note 69).
96
Megan Lucas et al.