Bio-Rad Bio-Plex® Assay Builder User Manual
Page 8
Prepare Coupled Beads
Protect the beads from light by covering the tubes with aluminum foil.
Keep all tubes on ice until ready to use. Coupled beads must be mixed
manually prior to use when combining singleplex or premixed assays.
1.
Vortex the coupled beads (50x) at medium speed for 5 sec.
2.
Prepare a sufficient volume of coupled beads (1x) using
wash buffer
.
When preparing a multiplex assay, use equal volumes of each bead
(see sample below). Each well requires 1 µl of coupled beads (50x) for
each target adjusted to a final volume of 50 µl (refer to the table
below). These calculations can be done on the worksheet.
11
Volume of Coupled Beads (50x) in Each Well
y
a
s
s
A
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m
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µ
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1
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F
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:
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1
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:
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%
5
2
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I
8
1
=
%
5
2
.
1
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3
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=
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3
2
:
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4
2
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ll
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:
s
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%
5
2
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I
4
2
=
%
5
2
.
1
x
s
ll
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w
0
3
=
s
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f
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b
m
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T
s
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0
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)
x
0
5
(
:
s
y
a
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x
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m
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P
=
x
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l
p
-
3
l
µ
3
2
)
8
3
p
,
a
-
B
k
I
,
2
K
R
E
(
:
s
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x
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µ
0
3
=
2
K
R
E
a
-
B
k
I
=
l
µ
0
3
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µ
0
3
=
8
3
p
=
l
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µ
0
9
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)
x
1
(
s
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3
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=
l
µ
0
5
x
l
µ
0
5
1
1
s
l
l
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w
0
3
=
l
µ
0
5
x
l
µ
0
0
5
1
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b
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µ
0
5
1
1
–
l
µ
3
2
=
l
µ
7
2
1
1
l
µ
0
0
5
1
–
l
µ
0
9
=
l
µ
0
1
4
1
Example Coupled Bead Calculations
Thaw Lysates
1. Retrieve the experiment lysates and lysates shipped with the assays
from –20ºC storage. These lysates were prepared using the protocol
in section 5 and contain 50% assay buffer.
NOTE: Refer to the table provided with the lysate packaging to
identify which lysates shipped with the assays (visit www.bio-
rad.com/products/phosphoproteins/ to download the PDF). Select
the treated and untreated lysates from the table, which is used to
determine the assay performance of each Bio-Plex phosphoprotein
and total target assay. These should not be considered as
references. Instead, activation signals and ratios should be based on
experimental control lysates. For determining total protein
concentrations, consider Bio-Rad’s
DC
protein assay kit (Bio-Rad
catalog #500-0112).
2.
Thaw the lysates at room temperature and then place them on ice.
3. If necessary, it is possible to further dilute the lysates. Lysing solution
freshly prepared (as specified in section 5) and assay buffer are
required. Use a 1:1 mixture of lysing solution and
assay buffer
to
further dilute the lysate.
10