Biology Reference
In-Depth Information
22.1.2
Method
22.1.2.1
Isolation of DIG fractions
Prepare 40
10
6
cells per group. For fewer cells, use less lysis buffer
to keep the cell concentration high.
Stimulate cells as appropriate.
The following steps must be performed on ice:
Prepare the buffers and sucrose mixtures (see
Section 1.1.3
).
Gently resuspend cell pellet in DIG lysis buffer. Lyse for 30 min.
Homogenize each lysate in a prechilled Dounce homogenizer (Wheaton;
7 mL glass tube with glass plunger) by applying 10 gentle strokes with the
loose-fitting plunger.
Mix homogenized lysate with an equal volume of 85% (w/v) sucrose and then
rotate at 4
C for 2 min.
Place this mixture at the bottom of a 14
10
6
to 100
95 mm Ultra-Clear ultracentrifuge
tube (Beckman). Slowly overlay this mixture with 5 mL of 35% sucrose
mixture. Next, carefully overlay the 35% layer with 5 mL of 5% sucrose
mixture. The overlays can be made in a variety of ways (e.g., Pasteur pipette,
pipette-aid set to “slow,” or a gradient-former apparatus), provided it is
performed carefully and consistently.
Centrifuge at 200,000
g
for 16-18 h at 4
C in a prechilled SW40 rotor.
Place tubes on ice, discard the upper 3-4 mL, then extract the opaque
band and the 35/5% interface (1 mL total), and label “DIG.” Discard
the next 5 mL, then extract the bottom 2 mL from the tube, and label
“Soluble.” Alternatively, the entire tube may be divided into 12
1mL
fractions. The extraction can be performed with a 1 mL pipetman or by
making a side puncture with a butterfly needle (this requires use of
polyallomer soft tubes).
Aliquot and add 4
sample buffer (at a 1:4 ratio), boil at 95
C for 5 min, and
Western blot directly or immunoprecipitate the fractions and then Western blot.
Alternatively, DIGs may be stored at
70
C. DIGs (but not the soluble fraction)
can be pelleted by diluting the fraction with an equal volume of MBS and then
centrifuging at 20,000
g
for 10 min at 4
C. Additionally, protein can be
precipitated and then assayed.
22.1.2.2
Immunoprecipitation of DIG fractions
Precoat protein A/G beads with 1
m
g antibody in 500
m
L phosphate-buffered
saline (PBS) for 2 h at 4
C.
Split the 1 mL DIG fractions into two portions of 500
L in microfuge
m
tubes.
Add 500
L MBS to each tube and then vortex.
Spin at 20,000
m
g
for 10-30 min at 4
C.
Remove and discard 950
L of the supernatant from each tube with a pipette
without disturbing the pellet.
m
