Two-Dimensional Polyacrylamide Gel Electrophoresis: A Separation Technique for Analysis of Proteins in Complex Protein Mixture Based on Charge and Molecular Weight
筆記録
For two-dimensional gel electrophoresis of protein mixture derived from homogenized tumor tissue, treat with buffer containing chaotropes, zwitterionic detergent, and reducing agent. Chaotropes and detergent denature and solubilize the proteins. Reducing agents reduce the proteins' disulfide bonds, generating free thiol groups and unfolding the proteins for optimal protein resolution.
Transfer the treated protein mixture into an immobilized pH gradient, IPG, strip holder. Place an IPG strip comprising linear pH gradient within the acrylamide gel matrix. Add mineral oil to prevent protein solution evaporation. Transfer the holder into an isoelectric focusing, IEF system. Allow the protein solution to rehydrate the strip.
Perform first-dimension IEF. The electric current causes charged proteins to migrate through the pH gradient towards oppositely-charged electrodes, immobilizing at their respective isoelectric points – pH at which the proteins have zero net charge.
Post-IEF, treat the IPG strip with sodium dodecyl sulfate, SDS, an anionic detergent that imparts negative charge to proteins. Equilibrate with an alkylating agent to modify the protein thiol groups, ensuring protein solubility during electrophoresis.
Load the strip onto a precast small-pore polyacrylamide-resolving gel and secure with molten agarose. Fill the chambers with SDS-containing electrophoresis buffer. Initiate second-dimension electrophoresis, perpendicular to IEF.
Negatively charged, larger molecular weight proteins move through the gel slowly towards the positively charged anode than smaller proteins, facilitating size-based separation.
Stain the gel. Proteins can be visualized as discrete spots on the gel.
