Question

Describe in some detail a western blotting and a ligand blotting technique for the detection and comparison of relative levels of a cellular protein (e.g. receptor) in two different cell lines.

Answer 1

Western Blotting- western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. In brief, the sample undergoes protein denaturation, followed by gel electrophoresis.

The blotting technique is a tool used in the identification of biomolecules such as DNA, mRNA, and protein during different stages of gene expression. Protein synthesis involves the expression of a DNA segment which gets converted to mRNA to produce the respective protein. Subtypes of blotting such as northern, western & southern depend upon the target molecule that is being sought. When a DNA sequence is a foundation or code for a protein molecule, the particular DNA molecule of interest can be blotted using Southern Blotting technique. During gene expression, when the DNA is expressed as mRNA for a protein production, this process can be identified by Northern blotting. Finally, the coded mRNA produces the concerned protein, this protein identification can be done by Western Blotting.THe priciple behind this is

The principle of the western blotting is based around a few broad steps: (a) the extraction of cellular proteins from a complex mixture of intracellular and extracellular proteins (from tissue, cells, etc.); (b) quantification of protein concentration and electrophoretic separation of proteins within a gel matrix; (c) transfer to a membrane with a high affinity for proteins; (d) “blocking” the membrane to reduce non‐specific binding; (e) antigen detection by antibodies specific for the protein(s) of interest; (f) incubation with a secondary antibody linked to a label (e.g., chemiluminescent or fluorescent); (g) development and detection of the signal, which is theoretically proportional to the degree of antigen/antibody binding; and (h) quantification of the resulting bands.

Ligand Blotting

The ligand blotting is a modification of western blot analysis, in which protein receptors, following electrophoretic separation and transfer onto a blotting membrane, are detected by the specific binding of labeled ligands. These are are subjected to one- or two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, after which the proteins are transferred to nitrocellulose paper. The paper is incubated with native LDL and then with an 125I-labeled antibody against LDL, and the bound antibody is visualized by autoradiography. The success of LDL blotting depends on the omission of sulfhydryl reducing agents from the electrophoresis system. Intrachain disulfide bonds allow the receptor to retain its binding activity even after electrophoresis in the presence of SDS. In identifying LDL receptors, the ligand blotting technique is as sensitive as immunoblotting with a monoclonal antibody against the LDL receptor; it can therefore be used to identify receptors when no anti-receptor antibodies are available. We use this technique to show that the LDL receptor of the rabbit adrenal gland has the same molecular weight as the LDL receptor of the bovine adrenal cortex and human fibroblasts. The ligand blotting technique may be generally applicable for visualization of other plasma membrane receptors after SDS-gel electrophoresis.