I mixed EDTA mouse plasma with standard gel sample buffer and got very smeary image on Western with no distinct bands. Is there something in the plasma that needs to be removed before I run gels?
Aarya Chitransh
Remove Lipids:Plasma contains lipids, which can cause smearing on the gel. To remove lipids, you can perform a quick spin or centrifugation step before loading the sample onto the gel. Centrifuging the plasma at a higher speed can pellet lipids, and the supernatant can be collected for further analysis. Deplete Albumin:Albumin, being a highly abundant protein in plasma, can sometimes dominate the gel and hinder the separation of other proteins. You may consider depleting albumin using methods such as albumin depletion columns or other commercially available kits. Precipitate Proteins:Precipitating proteins with a compatible reagent or using a protein precipitation method can help remove interfering substances and concentrate your protein of interest. Use Sample Buffer with Reducing Agent:Ensure that your sample buffer contains a reducing agent (e.g., DTT or β-mercaptoethanol) to break disulfide bonds and denature proteins. This is crucial for obtaining well-defined bands on Western blots. Properly Prepare the Gel:Make sure your gel is properly prepared and run under appropriate conditions. This includes using the correct percentage gel for the size range of your proteins and running the gel at a suitable voltage. Optimize Electrophoresis Conditions:Optimize electrophoresis conditions, such as voltage and run time, to achieve the best separation of proteins. Running the gel at a lower voltage for a longer time can sometimes improve resolution. Verify Protein Loading:Ensure that you are loading the appropriate amount of protein. Overloading can lead to smearing and poor band resolution. Use a Pre-cast Gel:Consider using pre-cast gels, as they are standardized and can provide better reproducibility. Check Antibodies and Detection System:Verify the specificity and sensitivity of your antibodies. Ensure that your detection system is optimized for the proteins of interest. Consider Gel Filtration or Size-Exclusion Chromatography:
When working with plasma samples for Western blotting, certain components in the plasma can interfere with electrophoresis and contribute to smearing on the gel or affect the separation of proteins. Here are some considerations and steps you can take to improve the resolution of your Western blot:
- If needed, you can consider using gel filtration or size-exclusion chromatography to separate proteins based on size before running them on a gel.
By addressing these considerations and optimizing your sample preparation and electrophoresis conditions, you should be able to achieve clearer and more distinct bands on your Western blot.
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