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The western blot (sometimes called the protein immunoblot) is a widely accepted analytical technique used to detect specific proteins in the given sample. It uses gel electrophoresis to separate native proteins or denatured proteins by its molecular weight. The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are stained with antibodies specific to the target protein.
Steps involved in Western blot #
1. Tissue Preparation #
Solid tissues are first broken down mechanically using a blender (for larger sample volumes), or homogenizer (smaller volumes), or by sonication. Assorted detergents, salts, and buffers may be employed to encourage lysis of cells followed to solubilize proteins.
2. Gel Electrophoresis #
The solubilized proteins are separated using SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) according to their electrophoretic mobility.
3. Transfer #
The proteins are transferred to a sheet of special blotting paper called nitrocellulose where proteins retain the same pattern of separation they had on the gel. In order to make the proteins accessible to antibody detection, they are moved from gel onto a membrane made of nitrocellulose or PVDF in a buffer containing Tris base and Glycine.
4. Staining (Optional) #
Protein gel (SDS-PAGE) has been stained with Coomassie Blue or silver staining in order to check the transfer efficiency.
5. Blocking #
Primary and secondary antibody (generally between 0.5 and 5 micrograms/mL) is added to this step. Blocking of non-specific binding proteins is achieved by placing the membrane in a dilute solution of 3-5% Bovine serum albumin (BSA) or non-fat dry milk in Tris- Buffered Saline (TBS).
5. Washing #
Washing of membrane to remove those non- specific proteins and un bound antibodies will be done using 5% detergents such as Tween 20 or Triton X-100 in a TBS. Washing has been done in each blocking steps.
6. Detection #
Radioactive detection: Radioactive labels do not require enzyme substrates, but rather allow the placement of medical X-ray film directly against the western blot which develops as it is exposed to the label and creates dark regions which correspond to the protein bands of interest.
Fluorescent detection: The fluorescently labeled probe is excited by light and the emission of the excitation is then detected by a photosensor such as CCD camera equipped with appropriate emission filters which captures a digital image of the western blot and allows further data analysis such as molecular weight analysis and a quantitative western blot analysis.
Sambrook J, Russell DW, (2001), Molecular Cloning: A laboratory manual, 3rd ed, Cold Spring Harbor Laboratory Press, New York.