Bio-Rad Immun-Blot® Opti-4CN™ Colorimetric Kits - Manual (Page 5)

Solutions for Nylon Membranes
1. Borate-Tween, (0.05 M Na
2
B
4
O
7
•10 H
2
O, 0.5 M NaCl,
1 x BT, 2 L : 5.0% MPO, 0.2% Tween-20, pH 9.3)
Dissolve 38.14 g NaH
2
B
4
O
7
•10 H
2
O and
58.44 g NaCl in 1.9 L dd H
2
O. Add
100 ml MPO and 4 ml Tween-20, then
bring solution volume to 2 L with dis-
tilled, deionized water (dd H
2
O) and mix.
2. Tris buffered saline, (0.02 M Tris, 0.5 M NaCl)
1 x TBS, 2 L: Add 4.84 g Tris base to 58.44 g NaCl
and bring to 1.8 liters with dd H
2
O.
Adjust to pH 7.5 with HCl. Add dd H
2
O
to a final volume of 2 L.
3. Tris-Tween-20 solu- (0.02 M Tris, 0.5 M NaCl, 0.2%
tion, 1 x TTBS, 1 L: Tween-20, 5.0% MPO)
Add 2 ml Tween-20 and 50 ml MPO to
1 L of TBS (above).
4. Avidin-HRP solu- Add 100 µl of Avidin-HRP to 100 ml
tion, 100 ml: of TTBS.
5. Phosphate buffered (0.01 M phosphate buffer, pH 7.2)
saline, 1 x PBS, Add 0.105 g sodium monobasic phos-
300 ml: phate (NaH
2
PO
4
•H
2
O), 0.600 g sodi-
um dibasic phosphate heptahydrate
(Na
2
HPO
4
•7 H
2
O), and 2.550 g sodium
chloride to 250 ml dd H
2
O. Adjust to
pH 7.2 with HCl and bring to 300 ml
with dd H
2
O.
6. HRP color develop- a. Dissolve 60 mg HRP Color
ment solution, Development Reagent in 20 ml ice
120 ml: cold methanol. Protect from light.
Make fresh daily.
b. Immediately prior to use, add 60 µl of
ice cold 30% H
2
O
2
(hydrogen perox-
ide, stabilized) to 100 ml room tem-
perature TBS. Mix this with (a)
above. Use immediately. This will
produce a 0.015% H
2
O
2
development
solution.
5
Solutions for Nitrocellulose Membranes
1. Borate-Tween solu- (0.05 M Na
2
B
4
O
7
•10 H
2
O, 0.2%
tion, 1 x BT, 2L: Tween-20 pH 9.3)
Dissolve 38.14 g Na
2
B
4
O
7
•10 H
2
O in
1.9 L distilled, deionized water (dd H
2
0).
Add 4 ml Tween-20, bring solution vol-
ume to 2 L with dd H
2
0 and mix.
2. Tris buffered saline, (0.02 M Tris, 0.5 M NaCl)
1 x TBS, 2 L: Add 4.84 g Tris base to 58.44 g NaCl
and bring to 1.8 liters with dd H
2
O.
Adjust to pH 7.5 with HCl. Add dd H
2
O
to a final volume of 2 L.
3. Tris Tween-20 solu- (0.02 M Tris, 0.5 M NaCl, 0.2%
tion, 1 x TTBS, 1 L: Tween-20)
Add 2 ml Tween-20 to 1 L of TBS
(above).
4. Avidin-HRP, 100ml: Add 100 µl of Avidin-HRP to 100 ml of
TTBS.
5. Phosphate buffered (0.01 M phosphate buffer, pH 7.2)
saline, 1 x PBS, Add 0.105 g sodium monobasic phos-
300 ml: phate (NaH
2
PO
4
•H
2
O), 0.600 g sodi-
um dibasic phosphate heptahydrate
(Na
2
HPO
4
•7 H
2
O), and 2.550 g sodium
chloride to 250 ml dd H
2
O. Adjust to
pH 7.2 with HCl and bring to 300 ml
with dd H
2
O.
6. HRP color develop- a. Dissolve 60 mg HRP Color
ment solution, Development Reagent in 20 ml ice
120 ml: cold methanol. Protect from light.
Make fresh daily.
b. Immediately prior to use, add 60 µl of
ice cold 30% H
2
O
2
(hydrogen perox-
ide, stabilized) to 100 ml room tem-
perature TBS. Mix this with (a)
above. Use immediately. This will
produce a 0.015% H
2
O
2
development
solution.
4
4006143B 8/27/98 08:44 AM Page 4