The fluorescence intensity of prestained color protein markers is a key indicator of their effectiveness, directly impacting the accuracy and reproducibility of experimental results. Its fluorescence intensity is regulated by multiple factors, encompassing the marker's inherent properties, experimental conditions, and environmental parameters. This requires a systematic analysis from molecular mechanisms to macroscopic operations.
The fluorescence intensity of prestained color protein markers primarily depends on the marker's chemical structure and stability. Prestained markers typically consist of a fluorescent dye covalently bound to a protein, and the dye's molecular structure directly influences its fluorescence properties. For example, rhodamine dyes, due to their rigid planar structure, offer high fluorescence efficiency and strong photostability. Fluorescein dyes, while offering high quantum yields, are susceptible to photobleaching, resulting in a significant decrease in fluorescence intensity under prolonged illumination. Furthermore, the binding efficiency of the dye to the protein is crucial. Improper or incomplete binding sites can result in insufficient exposure of the fluorophore or increased steric hindrance, thereby weakening the fluorescence signal.
Experimental conditions have a particularly significant impact on fluorescence intensity. Sample loading is a key factor. Excessive loading can lead to uneven protein diffusion in the gel, forming smears and reducing local fluorescence density. Insufficient loading can weaken the signal, making it difficult for the detection system to detect it. Electrophoresis conditions are equally important. Excessive voltage or prolonged electrophoresis time can increase the buffer temperature, accelerating the photolysis of the fluorescent dye and causing fluorescence intensity to decay over time. Furthermore, the gel concentration must be precisely selected based on the protein's molecular weight range. Too low a concentration can cause small proteins to smear, while too high a concentration can hinder the migration of large proteins, both of which can affect the clarity and intensity of the fluorescent bands.
The impact of environmental parameters on fluorescence intensity cannot be ignored. Temperature is a key factor. The fluorescence efficiency of fluorescent dyes generally decreases with increasing temperature. High temperatures intensify molecular thermal motion, leading to an increase in nonradiative transitions and a decrease in fluorescence quantum yield. Solvent polarity also significantly affects fluorescence properties. Polar solvents can alter the local environment of the fluorophore, resulting in a shift in emission wavelength or a change in fluorescence intensity. For example, some dyes exhibit high fluorescence intensity in hydrophobic environments, but this intensity may decrease in hydrophilic environments due to reduced solvation. Furthermore, the effect of solution pH on fluorescence intensity is specific. If the fluorescent group is a weak acid or base, changes in pH can alter its charge state, thereby affecting fluorescence efficiency.
The effects of protein properties on fluorescence intensity are complex. A protein's amino acid composition, charge, and post-modification status can alter its migration behavior in an electric field, affecting the position and intensity of the fluorescent band. For example, negatively charged proteins migrate faster during electrophoresis, potentially shifting the fluorescent band forward; glycosylation can increase the protein's molecular weight, slowing its migration rate. Furthermore, the hydrophobicity of a protein can affect its binding efficiency with the dye. More hydrophobic proteins may more easily form stable complexes with the dye, thereby enhancing the fluorescence signal.
The storage conditions of the marker play a crucial role in the long-term stability of fluorescence intensity. Prestained color protein markers should be protected from repeated freeze-thaw cycles, as this can disrupt the protein structure or cause dye shedding, reducing fluorescence intensity. Exposure to high temperatures or strong light can accelerate the photolysis of the dye, causing the fluorescence signal to gradually decay. Therefore, markers should be stored at low temperatures and protected from light, and the number of times they are opened should be minimized to maintain their fluorescence properties.
The compatibility of equipment and reagents is also a significant factor affecting fluorescence intensity. Air bubbles, abnormal electrode connections, or uneven temperatures in the electrophoresis tank can lead to uneven electric field distribution, affecting protein migration and the formation of fluorescent bands. Excessive methanol concentration in the transfer buffer can strip the dye from pre-stained proteins, causing bands to fade or disappear. Therefore, before the experiment, it is important to check the operating status of the equipment and prepare reagents strictly according to the instructions to ensure accurate fluorescence detection.
