Introduction

Polyacrylamide gel electrophoresis is widely used for separation, molecular weight determination, and compositional analysis of proteins and nucleic acids (1,2,3). Coomassie™ Brilliant Blue is arguably the most common organic dye applied to visualize proteins on gels following electrophoresis, as the staining procedure is easier and more rapid compared with other detection methods (4,5,6). The quest for more cost-effective densitometry methods led to investigations on the use of flatbed scanners over a decade ago (7,8). It is plausible that the apparent ambivalent conclusions drawn from these early investigations may have contributed to a lack of wider adoption of flatbed scanners for densitometry. Here, we consider the influence of light spectrum in densitometry, an important factor not addressed in previous reports (7,8). Using these findings, we proceed to demonstrate how reliable results can be consistently attained with flatbed scanner densitometry.

Materials and Methods

Protein Purification and Analysis

Protein molecular weight standards (Precision Plus Protein™ All Blue) and all reagents used for electrophoresis were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Recombinant His₆-tagged fusion protein (molecular weight, 29.4 kDa) was purified by immobilized metal affinity chromatography to >95% purity and quantified by the BCA™ assay (Pierce, Rockford, IL, USA). Vertical electrophoresis was carried out using a Mini-Protean® Electrophoresis Cell (Bio-Rad Laboratories), and known amounts of proteins (10–5000 ng/well) were resolved on a 16% discontinuous sodium dodecyl sulfate (SDS) polyacrylamide slab gel prepared using Laemmli's method (9). The polyacrylamide gels were stained overnight with colloidal Coomassie G-250 dye (Gelcode® Blue Stain Reagent; Pierce) and subjected to a Water Wash Enhancement™ Step (Pierce), where the stain was replaced with several changes of ultrapure water until a clear background was achieved. The destained gels were then dried between two sheets of cellophane using the GelAir™ drying system (Bio-Rad Laboratories).

Results and Discussion

Light Spectrum in Densitometry

Densitometric analysis into the extent of coloring by a dye provides a quantitative measure of the amount of protein present; wherein a monochromatic light beam scans both the area with and without the dye in stained gels for the respective intensities I(λ)and I_o(λ). Optical density at the wavelength λ is determined using the relation:

It is seldom appreciated that light is not ideally monochromatic (i.e., an impulse function in the spectral sense); nor is absorbance of a coomassie Blue-stained gel uniform over the visible spectrum. In addition, the photodetector used in any densitometer will not be able to produce signals that differentiate between light wavelengths. Hence, if the spectral distribution of both OD(λ) and I_o(λ) are known, one can essentially predict the measured optical density of any densitometer using the equation:

Using a stabilized 150 W halogen broadband light source, illumination was delivered through an optical fiber bundle to impinge the gel (Figure 1). Light transmitted through the gel was collected by a single fiber [numerical aperture (NA) of 0.48] with smaller diameter (1 mm) so that it harnesses light from the stained region alone. The spectral distribution of the light source without the stained gel was first recorded as a reference, followed by recording of the spectral distribution of the polyacrylamide gel with different protein masses prepared and stained with Coomassie Blue.

Figure 1. Experimental system used to determine spectral optical density distribution of a Coomassie Blue-stained polyacrylamide gel (Click to enlarge)

Computation with the reference yields spectral optical density distributions for differing amounts of protein (Figure 2). It can be seen that higher protein masses result in increased optical density values across the spectrum. The peak of each distribution (all approximately Gaussian) was consistently invariant at about the wavelength of 593 nm. It is noteworthy that at low amounts (e.g., 10 ng) of the protein, the profile and peak are almost nondiscernible (Figure 2). This is compatible with the binding characteristic of the colloidal Coomassie G-250 dye present in the Gelcode reagent, where the detection sensitivity for most proteins, as claimed by the manufacturer, is approximately 25 ng/band. It is also noteworthy that increases in optical density beyond 3400 ng/band protein is marginal and compatible with the known characteristic where complete binding of Coomassie G-250 dye to a protein (saturation) is approached (7). Clearly, the protein amounts falling within the linear concentration range is dependent on the type of protein that is present (10), and the 3400 ng/ band threshold appears to correspond reasonably with previous reports of the linearity response of 0–4000 ng/band for bovine serum albumin (BSA), smooth muscle myosin heavy chain, and actin (7). From each distribution and any light source with known spectral distribution, we are able to simulate the optical density expected using Equations 1 and 2.