Protein Protocols Protein Protocols

52 Root and Wang
Fig. 1. Representative plots of standard curve data. (A) Raw data for varying standard
protein mass per well.(B) Double-reciprocal plot of standard curve.
3. Methods
3.1. Microtiter Plate Assay
1. Adsorb the protein of interest to duplicate microtiter plates (see Note 1). Wash the
microtiter plate profusely with distilled water after the adsorption (see Note 2). One of the
microtiter plates is used for the kinetic silver staining assay and the other is for quantitative
ELISA or binding experiments. The microtiter plate for the kinetic silver staining
assay contains protein adsorbed to only a few of the wells (e.g., 4–16 wells).
2. Prepare a known concentration of the standard protein in distilled water (e.g., by dialysis)
for use in a standard curve (see Notes 2 and 3).
3. Apply varying concentrations (e.g., 50–1000 ng/mL) of the standard protein to the blank
wells on the microtiter plate at the same volume (50–200 µL) that was used to adsorb
protein in step 1. Cover the microtiter plate with a tissue to avoid dust from settling in the
wells and allow the protein to air dry for several days (see Note 4). Do not wash the plate
after this step!
4. Mix equal volumes of reagents A and B immediately before use. A fixed volume (e.g.,
100 µL) of the mixture is added quickly to the wells on the microtiter plate that were
adsorbed with protein in step 1, step 3, and to blank control wells. The time for the addition
of reagent to each well should be noted as time zero. All wells are filled in less than