In Western blotting (WB) experiments, prestained color protein markers act as "navigators" for molecular weight standards, providing researchers with a real-time and intuitive tool for band localization through dual labeling of color and molecular weight.
Prestained color protein markers consist of multiple standard proteins of known molecular weights covalently bound to specific dyes. Proteins of different molecular weights are labeled with different colors, forming a gradient of colored bands covering a specific molecular weight range. For example, one brand's marker contains 10 prestained bands, covering a molecular weight range of 10-180 kDa, with 70 kDa bands being orange-red, 10 kDa bands being green, and the remaining bands being blue or purple. This multi-color design allows researchers to quickly distinguish adjacent bands by color, avoiding misinterpretation caused by dense bands in single-color markers. During electrophoresis, the proteins in the marker separate according to molecular weight, forming colored "landmarks." Researchers can observe the position of the colored bands to judge the electrophoresis progress in real time, preventing bands from running off the gel or being incompletely separated.
The electrophoresis stage is the key step for prestained markers to play their localization role. Researchers must select a label with an appropriate range based on the molecular weight of the target protein, ensuring the target protein is within the label's coverage area. For example, when detecting a protein with a molecular weight of approximately 50 kDa, a label containing a 50 kDa band should be selected. During sample loading, the label is typically applied to the edge channels of the gel and electrophoresed simultaneously with the sample. After electrophoresis begins, researchers can observe the migration of the label bands through the electrophoresis tank glass: when low molecular weight bands (e.g., a 10 kDa green band) approach the bottom of the gel, it indicates that electrophoresis is nearing completion; if high molecular weight bands (e.g., a 180 kDa blue band) are still located in the upper part of the gel, it indicates insufficient electrophoresis time. By dynamically monitoring the position of the label bands, researchers can precisely control the timing of electrophoresis termination, ensuring optimal separation of the target protein.
During the transfer stage, the colored bands of the pre-stained label directly reflect the protein transfer efficiency. After transfer, the experimenter can judge the transfer effect by observing the integrity of the colored bands on the membrane without staining the membrane: if the marker band is complete and its position is consistent with that in the gel, the transfer is successful; if a large number of marker bands remain on the gel, it indicates insufficient transfer, and the transfer conditions need to be adjusted. In addition, the colored bands of the markers can also serve as a reference for the transfer direction, avoiding experimental failures caused by membrane reversal. For example, in one experiment, only low molecular weight marker bands appeared on the membrane after transfer, while high molecular weight bands were missing. Upon inspection, it was found that the membrane was reversibly assembled, resulting in the failure of high molecular weight proteins to transfer to the membrane.
After immunoblotting, the colored bands of the pre-stained markers can serve as a molecular weight reference, aiding in the localization and molecular weight estimation of the target protein. The experimenter can compare the target protein band with the nearest marker band on the membrane to preliminarily determine its molecular weight range. For example, if the target protein band is between 70 kDa (orange-red) and 55 kDa (blue), its molecular weight can be estimated to be approximately 60-65 kDa. For applications requiring precise molecular weight determination, calibration can be performed using non-prestained markers: first, determine the approximate range of the target protein using prestained markers, then establish a molecular weight standard curve within that range using non-prestained markers to calculate the precise molecular weight of the target protein.
The use of prestained color protein markers requires attention to operational details to ensure accurate localization. Experimenters should avoid heating or boiling the markers, as they are premixed with loading buffer, and heating may damage the protein structure and affect migration. Repeated freeze-thaw cycles should also be avoided to prevent breakage of the dye-protein coupling bonds, leading to band tailing or discoloration. Furthermore, different buffer systems (such as Tris-Glycine and Bis-Tris) may affect the migration characteristics of the marker bands; experimenters must select the appropriate marker type based on experimental conditions and verify its accuracy using non-prestained markers during initial use.
Prestained color protein markers provide a real-time, intuitive band localization tool for Western blotting experiments through dual identification of color and molecular weight. From monitoring the electrophoresis process to verifying transfer efficiency and estimating the target protein's molecular weight, its role is crucial throughout the entire experimental procedure. By appropriately selecting the type of marker, following standardized procedures, and paying attention to details, experimenters can fully leverage its positioning advantages and improve the reliability and repeatability of experimental results.
