Bio-Rad Immun-Blot® AP Colorimetric Kits - Manual (Page 6)

Working solution (based on 20 assays of 5 ml each):
Tris buffered saline (20 mM Tris, 500 mM NaCl, pH 7.5)
(TBS) Add 100 ml of 10x TBS to a 1 L bottle and quesce to
1.0 L with dd water. Label this bottle “TBS.”
Citrate buffered (20 mM citrate, 500 mM NaCl, pH 5.5)
saline (CBS) For the Protein G-HRP assay, add 100 ml of 3x CBS to
200 ml dd water. Label this solution “CBS.”
HRP Color The HRP color development buffer solution is a 10x
development buffer concentrate. It should be diluted with filtered, deionized
water. Dilute to a 1x working solution by adding 1 part
HRP color development buffer concentrate to 9 parts
filtered, deionized water. For example, to make 100 ml
of 1x HRP color development buffer, mix 90 ml of
filtered, deionized water with 10 ml of 10x HRP color
development buffer concentrate. Mix well. Store excess
solution at 4 °C.
Since the HRP color development buffer is better stored
for longer periods as a concentrate, make as much 1x
solution as is practical and needed for the planned
experiments.
AP Color The AP color development buffer solution is a 25x
development buffer concentrate. It should be diluted with filtered, deionized
water. Dilute to a 1x working solution by adding 1 part
AP color development buffer concentrate to 24 parts
filtered, deionized water. For example, to make 100 ml
of 1x AP color development buffer, mix 96 ml of
filtered, deionized water with 4 ml of 25x AP color
development concentrate. Mix well. Store excess
solution at 4 °C.
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2. Work in well-ventilated areas. Avoid inhalation of vapors when
handling solutions containing DMF, diethylene glycol, 4-chloro-1-
naphthol, and BCIP.
3. Do not mouth-pipet any solutions.
1.5 Solutions
The following solutions must be prepared for each kit. The working
solution volumes are based on 20 assays developing 0.75 x 9.2 cm
nitrocellulose strips. For each strip, the assay uses 5 ml of each working
solution per incubation step. It is advisable to use at least 0.5 ml of
solution per square centimeter (cm
2 ) of membrane. For best results, the
membrane must be completely covered with solution in all wash and
incubation steps. Larger volumes can be used for convenience, however
all volumes should be increased proportionately to insure that all kit
reagents are consumed at the same rate.
Stock solutions
Tris buffered saline, (200 mM Tris, 5 M NaCl, pH 7.5)
10x (10x TBS)
Color development HRP color development buffer, 10x
buffer AP color development buffer, 25x
Color development Ready to use as provided in the kit.
reagents
Citrate buffered With the Protein G-HRP assay kit, add the contents of
saline (CBS) the citrate buffered saline to a 1 L bottle, add 900 ml dd
water, agitate to dissolve, and quesce to 1.0 L with dd
water. Label this solution “3x CBS.” Store at 4 °C.
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