Most biological samples contain non-protein substances and/or contaminants that may interfere with the resolution of the electrophoretic separation. Considering the fact that cell lysates usually contain macromolecules and small ionic molecules which may interfere with the electrophoretic process, these agents should be eliminated prior to electrophoresis, especially if their amount exceeds the critical interference threshold.
How to Remove Interfering Agents from Protein Samples
What are some of the interfering agents of 2D electrophoresis and how do they affect the electrophoretic process? How can you successfully remove them from your protein samples? Here is a quick rundown of some of the most common contaminants that may affect the results of your experiment along with some suggestions on how you can take them out of your precious protein samples.
You need to keep in mind that proteins are the only analytes in 2D electrophoresis. As such, everything else that may be present in the lysate can be considered as contaminants or interfering agents. This includes polysaccharides, nucleic acids (DNA and RNA), lipids, phenolic compounds, small ionic molecules and albumin and IgG in human serum. Residual salts, buffers and detergents used during the extraction and solubilization of your protein samples can also be considered as interfering agents.
Polysaccharides. Since polysaccharides are composed of large molecules, these compounds can clog gel pores which may ultimately result in precipitation or extended focusing times. Some negatively charged polysaccharides can even bind with your proteins through electrostatic interactions and make it difficult for you to proceed with your experiment.
To remove these contaminants from your sample, you can use TCA, ammonium sulfate or phenol and ammonium acetate and then centrifuge it to precipitate the high molecular weight polysaccharides.
Nucleic acids. Nucleic acids can interfere with the results of your experiments in more ways than one. These compounds can clog gel pores, increase sample viscosity, and bind with your proteins through electrostatic interactions. They may also create background smears if the protein samples are visualized through silver staining.
Nucleic acids can best be removed from your sample through shearing or enzymatic digestion. As such, you can shear all those DNAs through sonication and/or digest them to mono- or oligonucleotides through the addition of protease-free DNase/RNase mixture.
Lipids. The presence of lipids can reduce the solubility and affect the molecular weight and isoelectric point (pl) of your protein sample. In addition, lipids can also form complexes with detergents and reduce their effectiveness as protein solubilizing agents. To remove lipids, use excess detergent during the solubilization phase or precipitate them using organic solvents.
Phenolic compounds. Phenolic compounds which are present in most plant tissues can modify proteins through oxidative reaction. To prevent this, you can use reductants such as DTT, B-mercaptoethanol, and ascorbate during extraction and separate them from the phenolic compounds through rapid precipitation. You should also use inhibitors such as diethyldithiocarbamic acid or thiourea to inactivate polyphenol oxidase and remove phenolic compounds by adding polyvinylpyrrolidone (PVP) or polyvinylpolypyrrolidone (PVPP) in the extraction solution.
Small ionic molecules. Negatively charged molecules such as nucleotides, phospholipids and metabolites may render your protein sample too conductive and may result in poor focusing. Apply TCA/acetone precipitation and dialysis methods to remove this type of contaminant.
Albumin and IgG. The high concentration of these two protein components in the human serum often masks the presence of other lower abundance proteins with similar molecular weight and isoelectric point. You can remove such contaminants by using affinity resins.
Salts, buffers and detergents. Residual salts in your sample may result in high strip conductivity, prolong the time required for IEF, initiate water movement and cause horizontal streaking. Strong ionic detergents also interfere with the electrophoretic process since they bind with the proteins and cause poor focusing.
To remove salt contaminants, you can apply dialysis, gel filtration or gel precipitation and resuspension methods. On the other hand, you can remove ionic detergents by diluting the SDS-containing sample in a rehydration solution containing a zwitterionic or nonionic detergent or by precipitating your protein sample in acetone.
G-Biosciences offers Perfect-FOCUS™ that removes the majority of interfering agents prior to 2D electrophoresis.
