18. Remove the aqueous upper layer without disturbing the interface. Note the volume of aqueous layer removed. To the phenol layer, add a volume of PBS equal to that removed. Rock at
RT for 4 h.
19. Repeat step 17 and remove aqueous upper layer.
20. Prepare dialysis tubing and transfer the final phenol layer and
interface to dialysis tubing (see Subheading 3.6, steps 18 and
19. Ensure chemical compatibility of the dialysis tubing type
prior to use with a phenol-saturated solution).
21. Place in a 7 L dialysis tank and dialyze for 48–72 h with
running deionized water. During dialysis, occasionally gently
knead the tubing to help break up large chunks of material,
being sure to wear gloves in order to prevent exposure to any
residual phenol.
22. Transfer the dialysis tubing to 7 L of pyrogen-free, 0.2μm
filtered water and dialyze at 4
C for 24 h.
23. Remove the samples from their dialysis membranes and transfer to clean sample tubes. Rinse the dialysis tubing with
pyrogen-free, 0.2μm filtered water to recover any particulate
material.
24. Make a homogeneous suspension of the material by breaking
apart large aggregates using bath sonication and/or manually
breaking using a cell scraper.
3.11 Partitioning
of Hydrophobic
and Hydrophilic
Proteins Using
Ammonium Sulfate
1. Determine the volume of your soluble protein mixture. Go to
http://www.encorbio.com/protocols/AM-SO4.htm,
an
online calculator, and enter in the starting volume, temperature
(which will be 4
C), desired ammonium sulfate 40%, and
starting ammonium sulfate 0%. The website will calculate the
amount of ammonium sulfate that needs to be added (see Note
73).
2. Add a stir bar to the protein solution, place it on a stir plate, and
begin stirring. Gradually add the ammonium sulfate crystals
and stir until completely dissolved. Transfer sample to 4
C (see
Note 74).
3. Incubate the sample at 4
C for 4–16 h; be sure sample is
stirring at all times (see Note 75).
4. Remove the stir bar. Divide the protein solution equally among
centrifuge bottles and balance. Centrifuge at 27,000 Â g, 4
C
for 1 h.
5. Decant the supernatant into a clean container. Store the pellet
at 4
C. This pellet is the 40% ammonium sulfate cut.
6. Determine the volume of the supernatant and repeat the online
calculation as in step 1, entering the new volume, desired
ammonium sulfate 50%, and starting ammonium sulfate 40%.
98
Megan Lucas et al.
RT for 4 h.
19. Repeat step 17 and remove aqueous upper layer.
20. Prepare dialysis tubing and transfer the final phenol layer and
interface to dialysis tubing (see Subheading 3.6, steps 18 and
19. Ensure chemical compatibility of the dialysis tubing type
prior to use with a phenol-saturated solution).
21. Place in a 7 L dialysis tank and dialyze for 48–72 h with
running deionized water. During dialysis, occasionally gently
knead the tubing to help break up large chunks of material,
being sure to wear gloves in order to prevent exposure to any
residual phenol.
22. Transfer the dialysis tubing to 7 L of pyrogen-free, 0.2μm
filtered water and dialyze at 4
C for 24 h.
23. Remove the samples from their dialysis membranes and transfer to clean sample tubes. Rinse the dialysis tubing with
pyrogen-free, 0.2μm filtered water to recover any particulate
material.
24. Make a homogeneous suspension of the material by breaking
apart large aggregates using bath sonication and/or manually
breaking using a cell scraper.
3.11 Partitioning
of Hydrophobic
and Hydrophilic
Proteins Using
Ammonium Sulfate
1. Determine the volume of your soluble protein mixture. Go to
http://www.encorbio.com/protocols/AM-SO4.htm,
an
online calculator, and enter in the starting volume, temperature
(which will be 4
C), desired ammonium sulfate 40%, and
starting ammonium sulfate 0%. The website will calculate the
amount of ammonium sulfate that needs to be added (see Note
73).
2. Add a stir bar to the protein solution, place it on a stir plate, and
begin stirring. Gradually add the ammonium sulfate crystals
and stir until completely dissolved. Transfer sample to 4
C (see
Note 74).
3. Incubate the sample at 4
C for 4–16 h; be sure sample is
stirring at all times (see Note 75).
4. Remove the stir bar. Divide the protein solution equally among
centrifuge bottles and balance. Centrifuge at 27,000 Â g, 4
C
for 1 h.
5. Decant the supernatant into a clean container. Store the pellet
at 4
C. This pellet is the 40% ammonium sulfate cut.
6. Determine the volume of the supernatant and repeat the online
calculation as in step 1, entering the new volume, desired
ammonium sulfate 50%, and starting ammonium sulfate 40%.
98
Megan Lucas et al.