In a previous post we provided you with five great tips for researchers working with proteins. Today we will present you with five more tips which are sure to improve the quality of your proteomic experiments.
- Negatively charged polysaccharides that contain sialic acid can produce horizontal streaks similar to those generated by nucleic acid contaminants. Ultracentrifugation is often sufficient to remove carbohydrates from samples.
- To prevent vertical streaking, limit the amount of protein added onto an IPG strip. Compensate for such decreases in sample load by using a more sensitive staining technique, such as silver staining.
- Reusing electrophoresis running buffer can result in poor separation and vertical streaking due to the depletion of ions and SDS in the running buffer. Avoid this practice, especially if vertical streaking is a persistent problem.
- Vertical streaking on second-dimension gels is often caused by gaps between the IPG strips and the gels. Ensure the second-dimension gel has a straight and level top edge, and that the IPG strip is in direct contact with the gel along its entire length.
- If some of the bands on your gel are not staining or appear faint, use silver stain as usual, then agitate it slowly in deionized water for 30 minutes and repeat. Then apply the silver stain again, starting with the silver reagent step. Proteins that did not stain on the first cycle will stain to full intensity.
For more great tips visit www.expressionproteomics.com