Bio-Rad Biotinylated Standards - Manual (Page 6)

Section 5
Assay Procedure
5.1 General Recommendations
1. Solution volume. The liquid in the incubation vessel should be at least
0.25 cm deep to insure the membrane is completely submerged during
incubation. There should be at least 0.5 ml of reagent per cm
2 of mem-
brane. Larger volumes may be used for convenience.
2. Handling the membrane. Wear clean plastic gloves or use forceps to
avoid leaving fingerprints on the membrane.
3. Temperature. All steps are performed at room temperature (22-25 °C).
4. Incubation vessels. Incubation vessels made of plastic are preferred
since avidin binds to glass even in the presence of detergents.
Siliconized glass is acceptable.
5. Membrane incubation. Agitation with a rotating shaker platform
enhances incubation efficiency. If a shaker platform is not available,
hand mixing every few minutes and extended incubation periods will
suffice.
6. Detection. It is best to detect only one membrane per incubation ves-
sel. Should it become necessary to use more than one membrane per
incubation vessel, calculate the solution volume based on the mem-
brane surface area, not the vessel size.
5.2 Protocol for SDS-PAGE Electrophoresis
1. When using HRP conjugates, dilute standards 1:4 in sample buffer.
When using AP conjugates, dilute the standards 1:20 in sample buffer.
Heat for 5 min at 95 °C. Cool and load 10 µl/well for mini-gels with
1.5 mm thick, 10 well combs. Load 10-15 µl/well for full length gels
(16-20 cm) with 1.5 mm thick, 15 well combs. Optimal load volumes
will vary with well size and gel thickness. Serial dilutions are recom-
mended to determine optimal loading volumes.
2. Perform the SDS-PAGE electrophoresis under the conditions
described in the instrument instruction manual.
3. Electrophoretically transfer the proteins to nitrocellulose membrane
following the instructions provided in the Trans-Blot
® cell, Trans-Blot
SD cell or Mini-Trans-Blot cell manual.
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6. Second antibody, avidin-HRP or avidin-AP solution, 100 ml:
Dilute blotting grade second antibody HRP or AP conjugate 1:3,000 by
adding 33 µ l to 100 ml antibody buffer. Dilute avidin conjugate
1:3,000 by adding 33 µl to the same solution.
7. HRP color development solution*, 120 ml:
(4-chloro-1-naphthol, catalog number 170-6534)
a. Dissolve 60 mg of HRP color development reagent in 20 ml
methanol. Protect from light. Make fresh daily. Label this solution A.
b. Immediately prior to use, add 60 µl of 30% H
2
O
2
(hydrogen perox-
ide, stabilized) to 100 ml room temperature TBS. Mix this with solu-
tion A. Use immediately. Solution B must be at room temperature
before addition of solution A to prevent undesirable precipitates.
8. Ap color development buffer**, 1 liter:
100 mM Tris, 1 mM MgCl
2
, pH 9.5
Add 12.1 g Tris to 204 µl MgCl
2
solution (4.9 M MgCl
2
• 6H
2
O) in 1 L
dd H
2
O. Adjust to pH 9.5 with HCl.
9. AP color development solution**, 100 ml:
(BCIP, catalog number 170-6539, and NBT, catalog number 170-6532)
a. Dissolve 30 mg NBT and 15 mg BCIP in 1 ml DMF. Vortex until
solids are well suspended. Add 1 ml AP color development buffer.
Vortex until solids dissolve. Label this solution A.
b. Immediately prior to use, add solution A to 100 ml of room tem-
perature AP color development buffer. Use immediately. The final
concentrations should be 0.3 mg/ml NBT, and 0.15 mg/ml BCIP.
* *HRP Conjugate Substrate Kit (catalog number 170-6431) simplifies the
color development procedure. The kit contains premixed 4-chloro-1-
naphthol and hydrogen peroxide solutions, and preweighed 10 x color
development buffer, for easy preparation of color development solutions.
**AP Conjugate Substrate Kit (catalog number 170-6432) simplifies the
color development procedure. The kit contains premixed BCIP and NBT
solutions, and preweighed 10 x color development buffer, for easy prepa-
ration of color development solutions.
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