Bio-Rad Biotinylated Standards User Manual
Page 5
Section 4
Solutions
4.1 Solutions for Electrophoresis Gels
1.
Sample buffer (SDS-PAGE reducing buffer)
Distilled water
4.0 ml
0.5 M Tris-HCl pH 6.8
1.0 ml
Glycerol
0.8 ml
10% (w/v) SDS
1.6 ml
ß-mercaptoethanol
0.4 ml
0.1% (w/v) bromophenol blue
0.2 ml
8.0 ml
4.2 Solutions for Nitrocellulose Membranes
1.
TBS - Tris buffered saline, 2 liters
20 mM Tris, 500 mM NaCl
Add 4.84 g Tris base, 58.44 g NaCl to 1.8 liters dd H
2
O. Adjust to pH
7.5 with HCl. Add dd H
2
O to a final volume of 2 liters.
2.
TTBS - Tween-20, Tris buffered saline.
20 mM Tris, 500 mM NaCl, 0.05% Tween-20. Add 0.5 ml Tween-20
to 1 liter of TBS.
3.
Blocking solution, 100 ml:
3% gelatin in TBS. Add 3 g gelatin to 100 ml of TBS. Heat to 50 °C
with stirring to dissolve, then cool. A microwave oven will quickly sol-
ubilize the gelatin, but do not heat above 65 °C.
4.
Antibody buffer, 200 ml:
1% gelatin in TTBS. Add 2 g gelatin to 200 ml TTBS. Heat to 50 °C
with stirring to dissolve. Cool before adding antibody. 100 ml is used
for the first antibody solution and 100 ml is used for the conjugate
solution.
5.
First antibody solution, 100 ml:
Dilute first antibody to appropriate titer in at least 100 ml antibody
buffer.
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Volume
Biotinylated SDS-PAGE Standards: 250 µl
Avidin-HRP: 2 ml
Avidin-AP: 1 ml
Applications
Biotinylated SDS-PAGE Standards:
per kit
Dilute 1:4 for HRP color development, 60-100 applications.
Dilute 1:20 for AP color development, 300-500 applications.
Avidin-HRP: makes 6 liters, enough for 60 to 120 uses.
Avidin-AP: makes 3 liters, enough for 30 to 60 uses.
Storage
Biotinylated Standards: -20 °C
Avidin-HRP and Avidin-AP: The conjugates are shipped on dry
ice, and can be stored at -20 °C prior to opening. Once
thawed, store reagents at 4 °C. Repeated freeze-thaw cycles
will damage the reagents.
Shelf life
1 year, if stored correctly.
Note: Biotinylated standards may show extraneous bands when stained
with a total protein stain.
Section 3
Safety Instructions
Read the entire instruction manual before beginning the assay.
1.
Wear gloves and protective clothing, such as a laboratory coat and gog-
gles, when preparing and working with the solutions in the assay.
DMF, 4-chloro-1-naphthol, and BCIP can cause skin and eye irritation,
and contact should be avoided. In case of contact, immediately flush
the skin and eyes with copious amounts of water for at least 15 min-
utes, and remove contaminated clothing.
2.
Work in well-ventilated areas. Avoid inhalation of vapors when han-
dling solutions containing DMF, 4-chloro-1-naphthol and BCIP.
3.
Do not mouth-pipet any solutions.
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