Protein extraction and sample cleanup are the most important steps to ensure optimal resolution and reduce variability of your 2-D gels. 2-D PAGE success depends on sample purity.
Interfering substances that can negatively impact SDS-PAGE and 2-DGE include salts, detergents, denaturants, or organic solvents, so it is crucial to eliminate these contaminants prior to analysis. Highly viscous samples indicate high DNA and/or carbohydrate content, which may interfere with separations. In addition, solutions at extreme pH values (for example, fractions from ion exchange chromatography) diminish the separation power of most electrophoresis techniques.
This section provides tips for total protein extraction, removal of contaminants such as salt, lipids, detergents, phenolic compounds, nucleic acids, polysaccharides, and plant-specific compounds, and for disulfide bond reduction.
Related Topics: Cell Disruption, Protein Solubilization, and Protein Fractionation and Depletion.
Bio-Rad's protein sample preparation products may be used with virtually all sample types. These products are based on well-understood purification and fractionation principles, and may be applied individually or in combination for effective sample preparation.
Product Selection Guide (extraction and cleanup).
Bio-Rad offers ReadyPrep™ protein extraction kits and the MicroRotofor™ cell lysis kits, which provide cell lysis and protein extraction protocols that are tailored to the specific needs of different sample sources.
ReadyPrep Protein Extraction Kit (Total Protein) — includes a strongly chaotropic extraction solution containing the zwitterionic detergent ASB-14. It generates protein samples that can be applied directly to IEF and 2-D gel electrophoresis.
MicroRotofor Cell Lysis Kits — four kits tailored to different organisms are available. All four kits are based on the same chaotropic protein solubilization buffer (PSB), which contains nondetergent sulfobetaine 201 (NDSB 201) along with urea, thiourea, and CHAPS for effective solubilization of most proteins. The kits generate total protein samples that are ready to be applied to SDS-PAGE, IEF, and 2-D gel electrophoresis. Different sample types have different requirements for effective cell disruption, and all four kits combine PSB with other elements to accommodate these specific needs.
Commercially available kits designed for removal of salts, high-abundance proteins, and other contaminants incorporate procedures such as affinity and size exclusion chromatography to improve resolution of 2-D gels.
Salt Removal of salts reduces streaking and improves reproducibility of 2-D gels (see figure below). Use either buffer exchange (desalting) or protein precipitation (which can also help concentrate the sample if needed).
Methods to remove salts from sample:
Salt removal using the ReadyPrep 2-D cleanup kit. E. coli extracts containing 1 M NaCl were electrophoresed before and after treatment with the 2-D cleanup kit. The samples were focused using 11 cm ReadyStrip™ pH 3–10 IPG strips, then run on Criterion™ 8–16% Tris-HCl precast gels for the second dimension.
Bio-Rad offers specialized products such as ReadyPrep™ 2-D cleanup kit for the removal of ionic contaminants such as detergents, lipids, and phenolic compounds from protein samples, and Bio-Spin® and Micro Bio-Spin™ 6 Columns for the removal of salt and contaminants.
Lipids, Detergents, and Phenolic Compounds Ionic contaminants such as lipids, detergents, and phenolic compounds interfere with isoelectric focusing of proteins resulting in poor 2-D gel resolution and reproducibility.
Methods to remove lipids, detergents, and phenolic compounds from sample:
Detergent removal using the ReadyPrep 2-D cleanup kit. E. coli extracts containing 1% SDS were electrophoresed before and after treatment with the ReadyPrep 2-D cleanup kit. The samples were focused using 11 cm ReadyStrip pH 3–10 IPG strips, then run on Criterion 8–16% Tris-HCl precast gels for the second dimension.
Nucleic Acids The presence of nucleic acids, especially DNA, interferes with separation of proteins by IEF. Under denaturing conditions, DNA complexes are dissociated and markedly increase the viscosity of the solution, which inhibits protein entry and slows migration in the immobilized gradient gel (IPG). In addition, DNA binds to proteins in the sample and causes artifactual migration and streaking.
Methods to remove nucleic acids from sample:
Polysaccharides The presence of polysaccharides interferes with separation of proteins by IEF. The effects are similar to nucleic acids, but are far more difficult to remove from samples.
Methods to remove polysaccharides from sample:
Plant-Specific Compounds Plant-specific compounds such as tannins, lignins, and chlorophyll can cause severe disruption of electrophoresis, in particular IEF.
Methods to remove plant-specific compounds from sample:
Disulfide bond formation is problematic for basic proteins because of their increased rate of formation in an alkaline environment. Protein solubility is also limited, which can interfere with resolution. Moreover, many reducing agents become negatively charged during IEF and migrate off the IPG strip, thus allowing disulfide bonds to re-form. Reducing disulfide bonds ensures proper protein migration and often yields better resolution of protein spots, fewer streaks, and greater reproducibility in 2-D separations.
Reduction-Alkylation Reducing agents are commonly used during sample preparation to cleave disulfide bond crosslinks within proteins and between subunits. Reduction in disulfide bonds decreases streaking and improves reproducibility of 2-D gels (see figure below).
Disulfide bond removal using the ReadyPrep reduction-alkylation kit. Protein samples were reduced using either 50 mM DTT in rehydration/sample buffer or the reduction-alkylation kit. Both samples were applied by cup loading onto 11 cm ReadyStrip pH 7–10 IPG strips and focused for the first dimension, then run on Criterion 8–16% Tris-HCl precast gels for the second dimension.
Bio-Rad offers the following reducing and alkylating reagents:
TBP and iodoacetamide are provided as components of the ReadyPrep Reduction-Alkylation kit , which also provides a complete protocol for performing irreversible reduction and alkylation reactions.
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