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In previous posts we provided you with several great tips for proteomic researchers (see five great tips for researchers working with proteins and more great proteomic applications tips). Today we will present you with the final in the series of application tips which are sure to improve the quality of your proteomic experiments.

• To ensure proper and consistent visualization with a silver stain, use ultrapure water with all organic contaminants removed for the final rinse of your staining vessel. In addition, reserve that vessel exclusively for silver staining, and separate it from other glassware in your laboratory.

 

• Proteins must be soluble if they are to be separated and identified on 2-D gels. Protein insolubility (precipitation) leads to loss of sample spots and streaking on 2-D gels.

 

• Fractionation may improve your 2-D result by reducing sample complexity, improving the range of detection, and enriching low-abundance proteins. Fractionation can be performed according to many protein properties, including subcellular location, solubility, size, charge, and pI.

 

• Improve the resolution and reproducibility of 2-D gels by performing sample cleanup to remove salts, charged detergents, phenolics, lipids, sugars, and nucleic acids. Cleanup will also reduce disulfide bonds.

 

• NaCl increases conductivity, extends the time required for focusing, causes electroendosmosis, and results in uneven water distribution in the gel. In general, the tolerated concentrations of NaCl for proper in-gel rehydration and cup loading are 10 mM and 40 mM, respectively.

 

• Nucleic acids bind proteins through electrostatic interaction, thereby interfering with isoelectric focusing. Nucleic acids can also clog the pores of the acrylamide matrix. Remove nucleic acids with nucleases and ultracentrifugation in the presence of carrier ampholytes. In addition, benzonase can be used in a sample together with urea to remove DNA or RNA contamination.

 

• Insoluble material in a sample obstructs gel pores, resulting in poor focusing and severe streaking. Remove these materials by high-speed centrifugation (for example, 20,000 x g for 30 minutes at 20°C).

 

• To monitor the initial progress of the electrophoresis on the IPG strips, add up to 0.001% of Bromophenol Blue to both the rehydration and equilibration buffers.

For more great tips visit www.expressionproteomics.com

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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

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