Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.
Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.
1. Sample Preparation
The first step in Western blotting is sample preparation. To prepare samples for running a gel cells and tissues need to be lysed to release the proteins of interest.
2. SDS-PAGE
The second step in Western blotting is protein separation. Proteins are separated based on molecular weight.
3. Protein Transfer
Just as proteins with an electrical charge can be induced to travel through a gel in an electrical field, so can the proteins be transferred in an electrical field from the gel onto a membrane, being either PVDF or nitrocellulose.
4. Antibodies and Accessory Reagents
5. Detection
For HRP conjugating secondary antibodies, ECL is traditionally used. An excellent ECL reagent we offer is RapidStep. The advantages of RapidStep are that no mixing of luminal or enhancer is required. Simply spray the membrane a few times and develop using x-ray film. RapidStep offers low pictogram sensitivity as well as emitting light for up to 2 hours after the addition of substrate.
In this video presentation, we have highlighted the major steps in Western blotting which are sample preparation, SDS-PAGE, membrane blocking/probing with antibodies, and detection. For each step of the process, proper protocols and appropriate reagents should be used to attain high-quality results.
The authors have nothing to disclose.