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Question: I have quantified my protein and peptides samples using BCA Pierce Kit and applied 20 ug of protein per well to run the electrophoresis. The molecular marker worked very well but there were no bands from my samples. During the BCA quantification I even had to dilute the samples 'cause the stocks quantification was too high (more than 2000 ug/ml), beyond the method sensitivity. Staining with bromophenol blue (comassie). What could be happening with my gel? Any ideas? Do you think silver staining could work better? Protein samples were diluted in water. Answer: Bromophenol blue is a commonly used tracking dye in gel electrophoresis. It serves several purposes in the electrophoresis process: Sample loading and tracking: Bromophenol blue is added to the DNA or protein samples to provide visual tracking during gel electrophoresis. Its blue color allows researchers to monitor the migration progress of the sample through the gel. This helps to estimate the distance traveled by the sample and determine when to stop the electrophoresis. Monitoring front migration: Bromophenol blue also serves as a front migration marker, indicating the progress of the electrophoresis. It migrates ahead of the DNA or protein bands and can be used to estimate the distance migrated by the sample relative to the front. Assessing gel run completion: Bromophenol blue, being a smaller molecule, migrates faster than larger DNA or protein molecules. When the bromophenol blue has reached the desired position in the gel, it indicates that the electrophoresis run is complete, and the gel can be stopped. Visualization of gel loading: Bromophenol blue helps visualize the sample loading process. By adding it to the sample, researchers can easily observe the sample entering the wells or loading slots of the gel. This ensures accurate and consistent sample loading. It's important to note that bromophenol blue does not interact with the DNA or protein molecules being analyzed. It simply aids in visualizing and tracking the progress of the electrophoresis run. Coomassie dye, specifically Coomassie Brilliant Blue, is commonly used in gel electrophoresis for protein staining. It serves the following purposes: Protein visualization: Coomassie dye binds to proteins, resulting in a visible color change. After electrophoresis, the gel is typically stained with Coomassie dye to visualize the protein bands. Coomassie dye interacts with various amino acid residues in the proteins, particularly basic amino acids such as lysine and arginine. Quantification: Coomassie staining can also be used for protein quantification. The intensity of the stained protein bands can be compared to known standards or a reference sample to estimate the relative protein concentration. Band excision: Coomassie-stained protein bands of interest can be excised from the gel for further analysis, such as mass spectrometry for protein identification or enzymatic digestion for peptide analysis. It's worth noting that there are different variations of Coomassie dye, such as Coomassie Brilliant Blue R-250 and Coomassie Brilliant Blue G-250. These variations have slightly different chemical properties but are generally used for similar purposes in gel electrophoresis.
