Biology Reference
In-Depth Information
2. If required a phenol purifi cation can be performed after
this step.
(a) Add 3 volumes of sucrose buffer (0.9 M Sucrose, 0.1 M
Tris-HCl pH 8, 10 mM DTT, 1 % Protease inhibitor
cocktail) and mix vigorously by vortex.
(b) Add 300
L of phenol (equilibrated at pH 8) and vortex.
Incubate 10 min at room temperature in a shaker.
(c) Centrifuge 5 min >10,000 ×
g
at room temperature. After
centrifugation two clear layers with a sharp interphase
should be seen. Transfer supernatant (phenol, should be
green) to a new 2 mL tube. If there is some turbidity add
200
μ
L of 2:1 of sucrose buffer:phenol, vortex and centri-
fuge again.
(d) Re-extract the aqueous phase by adding 300
μ
L of phenol
to the original. Vortex thoroughly and centrifuge 5 min
>10,000 ×
g
at room temperature. Transfer supernatant to
the tube with the previous phenolic phases.
(e) Optional clean-up of the phenol phase: adding 1 volume
sucrose buffer, vortex, and centrifuge. Transfer upper
(phenolic) phase to a new tube. If a thick interphase is vis-
ible, repeat this step once.
(f) Precipitate the proteins by adding 2 volumes of 0.1 M
ammonium acetate in methanol. Incubate overnight at
−20 °C.
(g) Continue the protocol in
step 5
.
3. The resolubilization of the pellet is a critical step since the pro-
teins should be in an adequate concentration. Here we recom-
mend starting with the addition of a low volume of buffer,
20-30
μ
L. If the buffer become very viscous (you can invert
the tube and nothing is coming down), or there is some undis-
solved proteins more buffer can be added. Protein solubilisa-
tion takes time so we recommend wait at least 30 min before
starting pipetting or adding more buffer. Always try to dissolve
the protein clumps by pipetting before adding more buffer.
Use adequate pipette tips to avoid losses.
4. The pellet may vary in size because it contains some carryover
of contaminants that are not dissolved in the protein resolubi-
lization step so a big pellet is not a sign of poor resolubilization
or yield. Samples washed with phenol generally do not gener-
ate a noticeable pellet in this step.
5. A good protein quantitation is needed because this protocol is
very sensitive to protein amount. We regularly use BCA method
because it is robust and also compatible with detergents.
μ
6. We routinely fractionate the proteins into two fractions, but
for a higher resolution three fractions can be picked. In this
