Calibration of the molecular weight ranges corresponding to the different colored bands in a prestained color protein marker is a multi-step process that integrates protein purification, chemical conjugation, electrophoretic mobility analysis, and quality control. The core principle is to precisely map the colored bands to the molecular weight ranges by covalently binding highly purified recombinant proteins to specific dyes, combined with the mathematical relationship between molecular weight and mobility in electrophoresis experiments.
First, molecular weight calibration relies on highly purified recombinant proteins. Manufacturers select a series of recombinant proteins of known molecular weight as standards. These proteins undergo multiple chromatographic purification steps to ensure electrophoresis-grade purity (typically >95%). For example, a prestained color protein marker covering the 10-250 kDa range may contain 11 proteins. The molecular weight intervals must be designed to balance resolution of the low molecular weight region (e.g., 8 kDa) and visibility of the high molecular weight region (e.g., 250 kDa). The molecular weight accuracy of the purified proteins must be verified by mass spectrometry or amino acid sequencing, which is the first quality control step in the calibration process.
Second, chemical conjugation technology is key to imparting color to the protein bands. Dye molecules (such as Coomassie Brilliant Blue and fluorescein derivatives) covalently bind to lysine or cysteine residues on proteins, forming stable dye-protein complexes. The coupling reaction requires strict control of conditions (such as pH, temperature, and reaction time) to prevent changes in protein conformation or uneven dye binding. For example, some dyes tend to bind nonspecifically to proteins under alkaline conditions, resulting in biased electrophoretic mobility. Therefore, optimizing the reaction system (such as adding buffers or using mild coupling agents) is necessary to ensure binding specificity.
Electrophoretic mobility analysis is a core experimental step in calibration. In SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), the mobility of a protein is linearly related to the logarithm of its molecular weight. Manufacturers use unstained protein standards of known molecular weight (such as unstained recombinant proteins or native protein mixtures) to establish a mobility-molecular-weight standard curve. Prestained color protein markers are then electrophoresed under the same conditions. The molecular weight corresponding to each color band is determined by comparing the band position with the mobility of the unstained standard. For example, if a prestained band exhibits the same migration rate as a 100 kDa unstained standard during electrophoresis, it can be labeled as 100 kDa.
Color design must balance visualization and practicality. Different colored bands typically correspond to specific molecular weight ranges, such as green for low molecular weight (e.g., 10 kDa), red for mid-range molecular weight (e.g., 70 kDa), and blue for high molecular weight (e.g., 250 kDa). This design relies on the human eye's ability to distinguish colors, while also considering the dye's stability in the running buffer. For example, some dyes tend to fade easily under acidic conditions, while the cathode buffer for SDS-PAGE is typically slightly alkaline. Therefore, it is necessary to identify dyes that are stable in alkaline environments or to adjust the dye concentration (e.g., increasing the molar ratio of dye to protein) to compensate for potential fading.
Batch consistency is crucial for the calibration process. Manufacturers ensure consistent band position and color intensity across batches of prestained color protein markers through rigorous production processes (e.g., fixed raw material sources and standardized operating procedures) and quality control (e.g., comparing electropherograms of each batch). For example, a manufacturer may specify that the migration rate deviation for each batch of product must not exceed ±5%, otherwise the coupling conditions or purification process must be readjusted.
The compatibility of the user's experimental conditions is also a factor to consider during calibration. Different gel concentrations (e.g., 8%, 12%, 15%), running buffer formulations (e.g., Tris-Glycine, MOPS), or transfer conditions (e.g., voltage, time) may affect the mobility of prestained bands. Therefore, manufacturers typically provide recommended experimental conditions in the product datasheet and recommend that users use prestained color protein markers under these conditions to achieve the most accurate molecular weight calibration results.
Finally, the calibration of prestained color protein markers is a dynamic optimization process. With advances in electrophoresis technology (e.g., high-resolution gels, fast electrophoresis systems) and the diversification of user needs (e.g., wide molecular weight range, multicolor labeling), manufacturers continue to improve the design of prestained color protein markers, for example, by introducing new dyes to enhance band brightness or optimizing protein combinations to cover a wider molecular weight range, thereby providing users with more accurate and convenient molecular weight calibration tools.
