Protein Protocols Protein Protocols

384 Root and Wang
Fig. 1. Schematic diagram of the assembly of a microtiter plate for the copper iodide staining
assay. Avoid wells marked with “x” due to possible distortions from edge effects.
ing of bands can occur at high loads of protein with PVDF. Copper iodide staining of
silica leaves an uncharacteristic bluish background. Comparisons of the sensitivities of
copper iodide staining and SECS with other stains on a variety of common supports are
illustrated in Table 1.
4. Microtiter plate readers are to be avoided for quantitative measurements, because they are
usually not sensitive enough (copper iodide staining yields OD ≤ 0.1) and do not sample a
large enough area of the stained surface to detect any nonuniformity in the staining density.
5. Exceeding a staining time of 5 min can both damage nitrocellulose membranes and lead to
solubilization of adsorbed protein. A staining time of 2 min is optimal.
6. Washing of microtiter plates should be handled gently by dipping in beakers of deionized
water. Vigorous washing procedures often lead to nonuniform protein distribution and
consequent uneven staining of microtiter plates.
7. An example of a low cost densitometer is a desktop flatbed scanner, color Apple Macintosh
computer, and NIH Image software (public domain, by Wayne Rasband; further details
are available by personal communication).
8. SECS may be removed by concentrations of KI that are less than 2 M but will require
longer incubations (e.g., 90 min for 0.5 M KI).
9. The wells on the edge of microtiter plates should be avoided for quantitative measurements
because they tend to yield less accurate numbers.
10. Nitrocellulose quantitatively binds most proteins that are dot blotted onto it and retains
them well throughout copper iodide staining.