Detection is typically performed using chromogen or peroxide-linked secondary antibodies to catalyze a chromogenic or chemiluminescent reaction. The 2D gel pattern of rat liver microsomes shows more detail and sharper spot outlines when dissolved in SDS buffer with heating than in urea buffer and is better yet when. This technique exploits the specificity inherent in antigen-antibody recognition. Evidence is provided that 2D SDS PAGE is reproducible, robust and compatible with SDS in both dimensions including isoelectric focusing in tube gels, the first dimension. This is followed by probing with antibodies specific to the protein being studied on the membrane, a method that is similar to immunohistochemistry, but without a need for fixation. The membrane can then be blocked with serum albumin or milk solution to prevent non-specific antibody binding. This type of assay is more commonly selected than native gels due to its simplicity. The proteins are denatured during the process by a detergent called sodium dodecyl sulfate (SDS). Membranes can be of the nitrocellulose, polyvinylidene difluoride (PVDF), or nylon variety. An SDS-PAGE gel separates proteins based on their size. Protein binding to the membrane is an irreversible mechanism. As the proteins migrate out of the gel, they are captured on a membrane. In the electric field generated by a power supply, the proteins coated with negatively charged SDS migrate toward the positive electrode. After this, they are transferred to a synthetic membrane via dry, semi-dry, or wet blotting methods. Proteins are generally separated by size using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. This analytic technique proceeds in the following steps. Immunoblotting Procedures Figure: Immunoblot: proteins separated by molecular weight and represented by a dark band on a blot.
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