Protein Protocols Protein Protocols

1092 Stott
Fig. 2. Origin of light carryover between wells of an “opaque” microtiter plate.
carryover occurs via external reflection from the shiny top surface (Fig. 2). Carryover
is least for black plastic, although the loss of reflection within the well also reduces the
signal available to the detector. Multiwell strips have higher carryover along the strip
than between adjacent strips owing to the plastic web that links the wells. Similarly,
individual wells have lower carryover than joined ones.
Carryover varies considerably between instruments, but it is particularly low if the
instrument has an antireflection mask between the plate and detector. Individual instruments
should be assessed for carryover using the type of plates that will be used with it.
The pattern of carryover should be determined by reading light output from all wells of a
plate containing a single glowing well with a light output, which represents the highest
expected from the assay. The location of this well may affect the results, and carryover
should be assessed using each corner well and one close to the center of the plate.
2. Materials
1. High-quality deionized water (see Note 3).
2. Luminol stock solution: 1.25 mM luminol in 0.1 M Tris-HCl, pH 8.6. Store at 4°C in the
dark. Make up fresh each week. Luminol should be recrystallized as the sodium salt before
use (see Note 8).
3. Hydrogen peroxide: 30% (w/v). Store at 4°C.
4. p-Iodophenol stock solution: p-iodophenol, 1 mg/mL in dimethyl sulfoxide (DMSO).
Make up fresh each day.
5. Microfluor “B” microtiter plates (Dynatech Laboratories, Chantilly, VA).
6. Coating buffer: 0.1 M glycine, pH 8.8. Adjust pH using NaOH. Store at 4°C, and make up
fresh each week.
7. Rabbit anti-α fetoprotein (AFP) (Dako [Glostrup, Denmark] cat. no. A0008).
8. Phosphate-buffered saline (PBS), pH 7.2: 0.14 M NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8.1 mM Na2HPO4. Store at 4°C, and make fresh each week.
9. Blocking solution: PBS containing Bovine serum albumin (BSA) 0.1% (w/v). Make up
fresh each day.
10. PBS-Tween: PBS containing Tween-20, 0.05% (v/v). Make an additional batch using
high-quality deionized water (see Note 3). Store at 4°C, and make fresh each week.
11. Assay diluent: PBS-Tween containing BSA 0.5% (w/v). Make up fresh each day.
12. Working standards concentration range 0–800 ng/mL made fresh for each assay batch by
serial dilution of stock standard using normal human serum. Stock standard (1600 ng/mL)