> See also: > - [[SDS-PAGE]] # Western Blot > Southern (Edwin Southern) -> DNA > Northern -> RNA > Western -> Protein **Western blotting** (also known as *immunoblotting*) is an [[Antibody-Based Lab Techniques|antibody-based lab technique]] that allows researchers to identify a specific immobilized protein using an [[Antibodies|antigen specific antibody]]. | Component | Purpose | | -------------------------------------- | ------------------------------------------------------------- | | Western Transfer | Transfers the bands from the gel into the membrane | | Nitrocellulose (PVDF) | The membrane that binds to proteins | | Blocking Agent (Milk) | Blocks open sites on the membrane to prevent antibody binding | | Primary Antibody | Binds to target protein | | Secondary Antibody | Binds to the Fc of the primary antibody | | Tris Buffered Saline with Tween(TBS-T) | Removes nonspecific binding | | Conjugated Enzyme | Catalyzes a reaction to allow for band visualization | ### Western Transfer After a gel is run, it can be "imprinted" onto a special paper known as a **nitrocellulose membrane** - Its important to begin a transfer as soon as a gel is complete, as the samples can begin to diffuse through the gel matrix and cause the bands to become "puffier" if left sitting for too long (~20 min is too long) - After the transfer has been completed, the nitrocellulose membrane can be stored for long periods of time (over a year) Once a western ### Blocking ### Primary Antibodies ### Secondary Antibodies and Visualization **Nanobodies** are small, single-domain antibodies