Dissolution and reduction of samples for two-dimensional polyacrylamide gel electrophoresis
Dissolution and reduction of samples for two-dimensional polyacrylamide gel electrophoresis
Source : Proteomics: From Sequence to Function.
Operation method
basic program
Principle
Ideal 2-DE solubilization should be such that all non-covalently bound protein complexes and aggregates are disrupted to form a solubilized solution of the individual polypeptides. If this is not achieved, strongly bound protein complexes in the sample may cause new protein spots to appear in the 2-DE, with a corresponding decrease in the spot intensities indicating individual polypeptides. In addition, the solubilization method must allow for the removal of substances such as salts, lipids, polysaccharides, and nucleic acids that may interfere with 2-DE separation. Finally, the proteins in the sample must remain soluble during the 2-DE process.
Materials and Instruments
Protein Samples Move 1. Solubilization Sample solubilization is one of the most critical factors for the successful separation of proteins by 2-DE. The most commonly used 2-DE protein solubilization method is still a mixture of the following (so-called "lysis buffer"), 9.5 mol/L urea, 4% (m/V) NP40, 1% (m/V) dithiothreitol (DTT) as reducing agent, and 2% (m/V) SCA in the corresponding pH range. This formulation is suitable for many types of samples but is not a foolproof method, which was challenged by the extraction of membrane proteins, and the amphoteric decontaminant CHAPS was found to be effective in solubilizing membrane-damaged leukoplakia, especially at a concentration of 4% (m/V), when combined with a mixture of 2mol/L thiourea and 8mol/L urea. Thiourea is a more powerful denaturant than urea, but it cannot be used alone because of its poor water solubility, but its solubility is better in concentrated urea solutions, so urea-thiourea mixtures improve the ability to solubilize samples. Linear sulfotrimethylammonium ethylactone (sulphobetaine) detergents, such as SB3-10 or SB3-12, have also been seen to be effective solubilizing agents, but they are not compatible with high concentrations of urea. However, these reagents are not compatible with high concentrations of urea, which can be overcome when they are used at 2% (m/V) in combination with l mol/L urea, 2 mol/L thiourea, and 2% CHAPS. The detergent SDS disrupts most noncovalent protein interactions and is very effective in solubilizing membrane proteins, but the anionic nature of the detergent makes it impossible to use in IEF gels. However, SDS can be used in a pre-solubilization process prior to 2-DE in which the sample is first solubilized with l% (m/V) SDS and then diluted with a common solubilizing solution (e.g., lysate) in order to keep the protein in a solubilized state by displacing the SDS from the protein with a nonionic or amphoteric decontaminant. In order to effectively solubilize the sample and minimize the negative effects of SDS in the IEF, the ratio of protein to nonionic or amphoteric detergent (1:3) and SDS to nonionic or amphoteric detergent (1:8) must be carefully controlled. Nucleic acids can be problematic for the IEF because they can lead to an increase in the viscosity of the sample and, in some cases, can form complexes with the sample proteins, which can lead to artifactual migration and streaking. This can lead to artifactual migration and streaking. If these problems are suspected, it is best to add a suitably pure (protease-free) endonuclease to the sample solution to degrade the nucleic acids, or to utilize the ability of SCA to bind to nucleic acids and form complexes to remove the complexes by ultracentrifugation. 2. Reduction Protein disulfides are usually reduced by reagents containing free sulfhydryl groups such as DTT or β-mercaptoethanol, but reagents such as DTT are charged and therefore migrate to the outside of the gel during the IEF process, resulting in reoxidation of the sample proteins and loss of proteins solubility during the electrophoresis process. Recently, it has been reported that replacing sulfhydryl group-containing reagents with uncharged reagents such as tertiary butylphosphine has greatly increased the proteolysis ability in IEF, and has enabled 1D to 2D protein solubilization. It was recently reported that the replacement of sulfhydryl-containing reagents with an uncharged reducing agent such as tributylphosphine greatly enhanced the proteolysis in IEF and improved the efficiency of the 1D-2D transfer. For more product details, please visit Aladdin Scientific website.
Lysis Buffer