Western Blotting
Six steps to Western blotting
Step 1: Gel Electrophoresis
Step 2: Membrane Transfer
Step 3: Blocking
Step 4: Primary Antibody Incubation
Step 5: Secondary Antibody Incubation
Step 6: Western Blot Analysis
Step 1: Gel Electrophoresis
For most experiments, the first step to Western blot analysis is separating the proteins in a sample using polyacrylamide gel electrophoresis (PAGE). In SDS-PAGE, the proteins in the sample are coated with the detergent SDS. The proteins then migrate according to their size, with smaller proteins migrating more quickly through the gel. The percentage of polyacrylamide in the gel determines how easily proteins of various sizes can move through the gel. Higher percentage gels having a tighter gel matrix better for resolving smaller proteins.
Polyacrylamide gels may be purchased ready-to-use in a variety of percentages or gradients. You can also choose to hand cast your own gels to achieve a customized percentage.
What you'll need for gel electrophoresis:
- Azure Aqua Quad Mini-Cell: Designed for running 1–4 precast or handcast gels (cassette size 10cm x 8cm). The unit comes equipped with four gel casting stands and casting frames. This is perfect for handcasting your own gels.
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