Analysis of Gene Expression at the Protein Level

Analysis of Gene Expression‎ at the Protein Level (Western Blot) 1. Introduction - Proteins are translated from mRNA species and are considered to be the final products of gene expression‎. - To explore proteins, one key approach is to analyze them by electophoresis, a process in which a net charged molecule will move in an electric field. - SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), which developed in the mid-1960s, is a technique to separate protein according to their net charges, size, and shapes. - Western blot hybidization (Immunoblotting) is a procedure in which different types of proteins are separated by SDS-PAGE and immobilized onto a solid support, either PVDF or nitrocellulose membranes. - The protein of interest on the blotted membrane is then detected by incubating the membrane with a specific antibody as a probe. - The term western; this technique was developed after the Southern blot method and was named western blotting. - The western blot technique 1)is a sensitive, reliable and quantitative method that is widely employed in the analysis of proteins. 2)offer information about the species, size, and expression‎ levels. - Some general factors 1)The concentration of polyacrylamide gel Polyacrylamide % (w/v) Protein size (kDa) 7 20-200 10 12-65 11 8.0-35 14 5.0-27 16 3.0-15 2)PVDF membranes are durable and tear-resistant (recommend) 3)The specificity and activity of an antibody Monoclonal Ab : - raised in mice, has a single antibody specificity, a single affinity, and a single immunoglobulin isotype. - more specific than polyclonal antibodies. - are produced by a monoclonal population of cells that are derived form one cloned cells. Polyclonal Ab: - raised in rabbits; contains a variety of antibodies directed against the antigen of interest as well as nonspecific protein 2. Procedure 1)Separation of proteins by SDS-PAGE 2)Transfer of proteins onto a solid membrane 3)Incubation of the membrane with specific antibodies 4)Detection of hybridized signals 3. Principles 1)SDS is a strong, negatively charged detergent that is composed of a hydrophilic head and a long hydrophobic tail 2)SDS serves to denature proteins and leave them negatively charged. 3)SDS binds to hydrophobic regions of protein molecules and causes them to unfold into extended polypeptide chains. 4)As a results, the individual proteins are dissociated form other proteins and rendered freely soluble in the SDS solution 5)A reducing agent, b-mercaptoethanol (SH-CH2-CH2-OH), breaks any S-S bonds in proteins 6)During SDS-PAGE, the anions of SDS not only denature proteins but coat polypeptides at a ratio of about one SDS for every two amino acid residues; Each protein molecule binds large numbers of the negatively charged SDS molecules that overwhelm the protein’s intrinsic charge. 7)To enhance separation, polyacrylamide gels are used as supporting media ad molecular sieve 8)Molecular Weight (MW) : linear relationship between the log of the MW and its Rf. 9)Blotting (figures)