WESTERN BLOT Reagents: 2x SDS buffer Running buffer Transfer buffer Blocking buffer 1st and 2nd antibodies

What is a buffer? A buffer is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. HA + XOH → HOH (water) + XA (salt) Weak acid → conjugate base Its pH changes very little when a small amount of strong acid or base is added to it and thus it is used to prevent changes in the pH of a solution. Buffer solutions are used as a means of keeping pH at a nearly constant value in a wide variety of chemical applications. Many life forms thrive only in a relatively small pH range so they utilize a buffer solution to maintain a constant pH. One example of a buffer solution found in nature is blood.

Where are Proteins found in cells? Most proteins are found in the cytoplasm of the cell, however, some can be found inside the nucleus. Proteins are large biological molecules consisting of one or more chains of amino acids. Proteins perform a vast array of functions within living organisms, including catalyzing metabolic reactions, replicating DNA, responding to stimuli, and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in folding of the protein into a specific three-dimensional structure that determines its activity.

Lysis Buffer Different Lysis buffers are used to either lyse the cell membrane to release the protein in the cytoplasm or to lyse both the cell membrane and nuclear membrane to release proteins in the cytoplasm and inside the nucleus. The Lysis buffers used to lyse the cell membrane are gentle detergents, while those which lyse the nuclear membrane are strong detergents.

Running Buffer The running buffer provides a mobile path for the electrical current that carries the samples traveling through the gel. It contains very little SDS which gently coats the amino acid chains, allowing it to travel through the gel with the continuous electrical current.

Transfer Buffer Transfer buffers must enable both effective elution of proteins from the gel matrix and binding of the protein to the membrane. The choice of transfer buffer depends on the membrane being used and the physical characteristics of the protein of interest

Blocking Buffer Traditional protein blocking buffer contains BSA, casein and milk for Western blotting, ELISA and other immunoassay detection methods with micro plates and membranes. The protein blocking buffer is used to block off nonspecific binding sites so that the antibodies used is just specific to attach to the particular protein of interest.

1st and 2nd Antibodies 1st antibody is a protein which is specific to attach to the protein of interest from the cell. 2nd antibody is another protein which has a horse radish peroxidase enzyme that reacts with hydrogen peroxide to produce a chemiluminescence reaction that releases light, which in turn captured by a charged couple camera. The 2nd antibody is specific to the 1st antibody and the chemiluminescence reaction locates the location of the protein of interest on the membrane after the Western blot is run.

The Principles of Western blot The western blot (sometimes called the protein immunoblot) is a widely accepted analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide. The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are stained with antibodies specific to the target protein, which in turn give out light due to a chemiluminescence reaction for the detection of the location of that specific protein. The proteins are separated by size (in kD) compared to a colored marker on the gel.