Bio-Rad Bio-Plex® Assay Builder User Manual
Page 6
3.
Prepare an adequate volume of lysing solution (refer to the table on
the left). For 10 ml of lysing solution, add 40 µl of
factor 1
and
20 µl of
factor 2
to 9.9 ml of
cell lysis buffer
. Vortex gently to mix
and set aside on ice. Then add 40 µl of 500 mM PMSF.
4. Lyse the samples:
Adherent and Suspension Cells
a) Immediately add the lysing solution to the cells. The amount of
lysing solution needed depends on the cell concentration in the
culture vessel (see table on the left).
b) Agitate the cells as follows:
Culture Plate — For suspension cells, place the plate on ice and
pipet the contents of the wells up and down 5 times. For adherent
cells, scrape the cells with a cell scraper. For both, agitate the
plate on a microplate shaker at 300 rpm for 20 min at 4ºC.
Other Culture Vessel — Transfer the cell lysate to a centrifuge
tube and rotate for 20 min at 4ºC.
HINT: Freeze-thawing the lysate once using dry ice or a –20ºC
freezer may increase the extent of the lysis. Alternatively, briefly
sonicate (eg., with a Sonifier 450 as follows: Duty cycle = 40,
Output = 1, Pulse sonicating = 18 times).
c) Centrifuge the samples at 4,500 g for 20 min at 4ºC.
Tissue Samples
a) Immediately add 500 µl of lysing solution to the tissue grinder
and grind the tissue sample on ice using about 20 strokes.
b) Transfer the ground tissue to a clean microcentrifuge tube
and freeze the sample at –70ºC.
c) Thaw the samples, then sonicate on ice as suggested above.
d) Centrifuge the samples at 4,500 g for 4 min.
5.
Collect the supernatant without disturbing the pellet.
6.
Determine the lysate protein concentration. The protein
concentration should be 200–900 µg/ml. It may be necessary to
test-lyse your samples with different volumes of lysing solution to
obtain the specified protein concentration range.
6
Section 5
Lysate Preparation
This section provides instructions for preparing lysates derived from cell
culture and tissure samples. For optimal recovery and sensitivity with the
phospho-Histone H3 assay, refer to suggested protocol for lysate
preparation.
1.
Rinse the samples with
cell wash buffer
as follows:
Adherent Cells — Stop the treatment reaction by aspirating the
culture medium and quickly rinsing the cells with ice-cold cell
wash buffer. The volume of cell wash buffer required is the
same as the volume of aspirated cell culture medium. Keep the
cells on ice.
Suspension Cells — Stop the treatment reaction by adding ice-
cold wash buffer to the cells. The volume of cell wash buffer
required is twice that of the culture medium. Centrifuge the cells at
1,000 rpm for 5 min at 4ºC. Aspirate the supernatant.
Tissue Samples — Rinse the tissue sample with cell wash buffer
once. Cut the tissue into 3 x 3 mm pieces and transfer them to a
2 ml tissue grinder.
2.
Prepare 500 mM PMSF by dissolving 0.436 g PMSF in 5 ml DMSO.
Store as 0.5 ml aliquots at –20ºC. Aliquots can be frozen and
thawed up to 5 times.
e
r
u
t
l
u
C
l
e
s
s
e
v
m
u
i
d
e
m
e
r
u
t
l
u
C
e
m
u
l
o
v
n
o
i
t
u
l
o
s
g
n
i
s
y
L
e
m
u
l
o
v
s
e
t
o
N
e
t
a
l
p
ll
e
w
-
6
9
ll
e
w
/
l
µ
0
0
1
ll
e
w
/
l
µ
5
7
o
t
s
ll
e
c
w
o
r
G
e
c
n
e
u
lf
n
o
c
%
5
8
–
0
8
g
n
i
v
a
e
l
d
n
e
m
m
o
c
e
R
y
t
p
m
e
s
ll
e
w
l
a
n
r
e
t
x
e
t
c
e
f
f
e
e
g
d
e
o
t
e
u
d
h
s
i
d
e
r
u
t
l
u
c
m
c
0
1
l
m
0
1
l
m
3
–
2
o
t
s
ll
e
c
w
o
r
G
e
c
n
e
u
lf
n
o
c
%
0
9
–
0
8
Lysing Solution Volume Guide
7