Protein Protocols Protein Protocols

Kinetic Silver Staining of Proteins 53
10 min from the time of mixing reagents A and B; otherwise, excessive silver development
in the reagent may cause a high background.
5. Read optical densities of each well with a microtiter plate reader at 405 nm (see Note 5).
The time elapsed from time zero for each well to reach 0.25 OD (lag time) is noted as the
lag time.
6. The lag time is plotted against the mass per well of adsorbed protein in the standard curve
(step 3), which typically yields an inverse sigmoidal shape curve. Comparison of the lag
times of the sample wells to the standard curve allows the determination of the total mass
of protein in the sample well. The standard curve may be linearized to a sharply biphasic
shape curve by plotting 1/lag time vs 1/protein per well (see Fig. 1).
3.2. Cellulose Assay
1. Mix equal volumes of reagents A and B immediately before use.
2. Stain cellulose (see Note 6) with adsorbed proteins by immersion in the mixed reagents
for at least 1 h.
4. Notes
Table 1
Compounds Known to Interfere with Kinetic
Silver Staining
Compounds Concentration in the
staining reagent
TCA >0.01 mM
Glucose >1 mM
β-Mercaptoethanol >0.1 mM
DTT >0.01 mM
KOH >0.01 mM
EDTA >1 mM
Phosphate >0.01 mM
SDS > 1 mM
Triton X-100 >0.0005%
Ammonium sulfate >1 mM
Urea >1 mM
Imidazole >0.01 mM
Tris >1 mM
NaCl >0.01 mM
KCl >0.01 mM
Guanidine HCl >0.01 mM
1. The wells on the edge of microtiter plates should be avoided for quantitative measurements
because they tend to yield less accurate numbers.
2. Washing of microtiter plates is essential, as residual buffer reagents may interfere with
silver staining (see Table 1). The washing is performed by gently dipping in beakers of
deionized water. Vigorous washing was avoided for fear of losing adsorbed protein.
3. Kinetic silver staining shows little protein-to-protein variation (