Success or failure of any protein analysis depends on sample purity. Interfering substances that can negatively impact gel electrophoresis include salts, detergents, denaturants, or organic solvents (Evans et al. 2009). Bio-Rad products that can be used for contaminant removal are discussed in this section.
Related Sections: Cell Disruption, Protein Solubilization, and Sample Quantitation.
In addition to chemical contaminants, protein samples may contain an excess of biomolecules such as DNA or carbohydrates. Highly viscous samples indicate high DNA and/or carbohydrate content, which may also interfere with PAGE separations. In addition, solutions at extreme pH values, such as fractions from ion exchange chromatography, diminish the separation power of most electrophoresis techniques. Use one of the following methods as needed to remove these contaminants:
Bio-Rad offers kits designed for removal of salts, detergents, and other contaminants. They incorporate procedures such as precipitation and size exclusion chromatography to improve resolution of SDS-PAGE and 2-D gels. For contaminant removal Bio-Rad offers the following:
Bio-Rad products that can be used for contaminant removal. Top, Micro Bio-Spin and Bio-Spin columns; bottom, ReadyPrep 2-D cleanup kit.
Column Selection Guide
* 150 mM NaCl, 17.5 mM sodium citrate, pH 7.0 ** In Tris buffer. *** In RNase-free Tris buffer.
Fragment DNA with ultrasonic waves during cell lysis and protein solubilization
Add endonucleases like Benzonase
Precipitate protein with TCA/acetone (ReadyPrep 2-D cleanup kit) to diminish carbohydrate content
Evans DR et al. (2009). Concentration of proteins and removal of solutes. Methods Enzymol 463, 97–120.
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