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

1.3 Required Reagents Not Included
1. Sodium borate, 10-hydrate (Na
2
B
4
O
7
•10 H
2
O)–ACS reagent
grade.
2. Sodium chloride (NaCl)–ACS reagent grade.
3. Methanol–ACS reagent grade.
4. Hydrogen peroxide–30% H
2
O
2
aqueous solution, stabilized,
500 ml (J. T. Baker, catalog number 2186).
5. 1-methyl-2-pyrrolidinone (MPO)–ACS reagent grade
(for nylon membranes only).
1.4 Solutions
It is advisable to use the following solutions in volumes of
approximately 0.5 ml reagent per cm 2 of membrane. Larger
volumes may be used for convenience. The solution should be at
least 0.25 cm deep to insure that the membrane is completely
submerged during incubation. A 14 cm petri dish will hold one
3 x 5 cm rectangle, a 20 x 20 cm dish will hold one 15 x 15 cm
sheet.
The solution volumes that follow are recommended for a typi-
cal assay of one 15 x15 cm nitrocellulose or nylon sheet assayed
in a 20 x 20 cm incubation vessel, which requires 100 ml of solu-
tion to obtain the required depth of 0.25 cm. Adjust volumes for
the incubation vessel and the number of membranes being stained.
It is best to stain only one membrane per incubation vessel.
Should it become necessary to use one vessel for more than one
membrane, calculate the solution volume based on the membrane
surface area to be stained, not the vessel size.
3
1.1 Materials Provided
Catalog Quantity per
Number Product Description Package
170-6518 Avidin-Horseradish Peroxidase Conjugate 2 ml
(Avidin-HRP)
and
170-6529 N-Hydroxysuccinimide Biotinate in 4 ml
dimethylformamide (NHS-Biotin), 75 mM
170-6531 Tween-20, EIA grade 100 ml
161-0715 Tris 100 g
170-6534 HRP Color Development Reagent 5 g
(containing 4-chloro-1 naphthol)
The Biotin-Blot Protein Detection Kit contains enough NHS-biotin and Avidin-
HRP reagents to produce 2 liters of each working solution. This is sufficient to
assay approximately 500 membrane strips (0.6–0.8 x 10–15 cm size) or 25 mem-
brane sheets (15 x 15 cm size).
1.2 Storage and Stability
The components of the Biotin-Blot Protein Detection Kit
should be stored as follows:
Temperature Shelf Life
Avidin-HRP 4 °C 1 year
NHS-Biotin -20 °C 1 year
Tween-20 23–25 °C >1 year
Tris Base 23–25 °C >1 year
HRP Color Development Reagent* -20 °C 1 year
* Note: The HRP Color Development Reagent should be stored at
-20 °C, desiccated, and away from light.
2
4006143B 8/27/98 08:44 AM Page 2