I have been trying to visualize supercomplexes in BN PAGE, however my results are inconsistent. All the single complexes are visible, but supercomplexes are not. I am using an imidazole based buffer system and handcasting 3-13% gradient gels with a pipette. I have tried titrating digitonin. Suggestions on how to improve supercomplex visualisation will be helpful.
Noor Rajeeb
Sample Preparation: Ensure that your mitochondrial samples are of high quality and integrity. Optimize the isolation procedure to minimize degradation and maintain the integrity of the supercomplexes. Avoid overextraction or harsh conditions that could disrupt the native structure. Protein Extraction: Optimize the solubilization step to effectively solubilize the mitochondrial membranes and extract the supercomplexes. Use mild detergents such as digitonin, dodecyl maltoside (DDM), or n-dodecyl-β-D-maltoside (DM) for solubilization. Titrate the detergent concentration to achieve efficient solubilization without excessive disruption of the supercomplexes. Buffer System: Evaluate different buffer systems to optimize the separation and resolution of supercomplexes. Imidazole-based buffer systems, such as the one you mentioned, are commonly used in BN-PAGE. However, other buffer systems like Bis-Tris or Tricine may also be effective. Test different buffer compositions, pH values, and ionic strengths to optimize the migration and resolution of supercomplexes. Gel Composition: Experiment with different acrylamide concentrations and gradients in your gels. A 3-13% gradient gel should be suitable for resolving a range of protein sizes, but you may need to adjust the gradient or acrylamide concentrations for specific supercomplexes. Consider using precast gels for consistency and reproducibility. Electrophoresis Conditions: Optimize the electrophoresis conditions such as voltage, running time, and cooling. Run the gels at a constant voltage with appropriate cooling to minimize heat generation and prevent protein denaturation. Protein Loading: Optimize the protein loading amount to ensure sufficient protein concentration for detection without overloading the gel. Supercomplexes are typically present in relatively low abundance, so a higher protein concentration may be required to visualize them. Concentrate your samples if necessary using methods such as ultrafiltration or precipitation. Staining and Detection: Use sensitive protein staining methods suitable for BN-PAGE, such as Coomassie Brilliant Blue or silver staining. Consider fluorescent stains or antibody-based detection for increased sensitivity. Optimize the staining and destaining protocols to achieve clear visualization of the supercomplexes. Positive Controls: Include positive controls, such as purified supercomplexes or mitochondria from well-characterized sources, to verify the effectiveness of your BN-PAGE setup and detection methods.
Mitochondrial supercomplexes can be visualized using (BN-PAGE), although their detection can sometimes be challenging. Here are some suggestions to improve the visualization of mitochondrial supercomplexes:
By carefully optimizing these parameters, you can enhance the visualization of mitochondrial supercomplexes in BN-PAGE and improve the consistency of your results
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