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SDS-PAGE and Western blot are common immunoassay techniques employed to detect specific proteins. These techniques are especially useful in cancer studies to identify the upregulation or downregulation of proteins in the course of cancer.
188.8.131.52 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
SDS-PAGE developed by Laemmli (Renart, et al., 1996), is a relatively simple, rapid, and sensitive tool used to study the properties of proteins (Shi & Jackowski, 1998). It enables the separation of proteins according to their respective molecular weights on the basis that charged molecules migrate through a matrix in the presence of an electric field (Shi & Jackowski, 1998). The common support matrices used are agarose and polyacrylamide gel. Since proteins generally have smaller molecular weights compared to macromolecules such as nucleic acids, polyacrylamide gels are preferred (Shi & Jackowski, 1998).
Polyacrylamide gels are formed by cross-linking acrylamide chains with N,N2-methylene-bis-acrylamide (bis) (Choe & Cho, 1994). It is triggered by either chemical or photochemical systems (Shi & Jackowski, 1998). Chemical polymerization is brought about by N,N,N2,N2-tetramethylethylenediamine (TEMED) and ammonium persulfate (APS) (Shi & Jackowski, 1998). Photochemical polymerization is caused by riboflavin or methylene blue (Shi & Jackowski, 1998).
In chemical polymerization, APS dissolves in water to form persulfate free radicals which activate the acrylamide monomers (Shi & Jackowski, 1998). The activated acrylamide monomers now react with inactivated monomers to produce a long polymer chain (Shi & Jackowski, 1998). Due to its ability to carry electrons, TEMED then catalyses the polymerization reaction (Shi & Jackowski, 1998). Bis finally cross-links the elongating acrylamide chains to form the polyacrylamide gel (Shi & Jackowski, 1998). The resulting gel thus contains pores through which proteins migrate and are resolved (Choe & Cho, 1994).
Proteins are first concentrated in a large-pored stacking gel followed by their separation through a small-pored resolving gel based on a two-buffer system. Among the many different buffer systems developed, the most commonly used is that established by Orenstein and Davis in 1964 (Choe & Cho, 1994). This system utilizes glycine (pKa 9.6) in the running buffer, and Tris-HCl adjusted to pH 6.8 and pH 8.8 [BIOCHEMICAL TECNINQUES] in the stacking gel buffer and the resolving gel buffer respectively (Choe & Cho, 1994).
SDS is an anionic detergent used in SDS-PAGE to aid in the restriction of protein mobility based solely on the proteins' molecular weights (Choe & Cho, 1994). This results from the binding of proteins to SDS, which brings about conformational changes and denaturation of the proteins (Choe & Cho, 1994).
Upon the application of an electric field,protein molecules are packed into a region between leading chloride ions and trailing glycinate ions (Choe & Cho, 1994). As the proteins migrate into the resolving gel, the higher pH causes the mobility of glycinate ions to surpass that of the proteins, bringing about their separation based on their molecular weights (Choe & Cho, 1994).
184.108.40.206 Wet Transfer
Although proteins are well-separated via SDS-PAGE, the polyacrylamide gel does not favour immunoblotting techniques due to its poor suitability to detect and characterize separated proteins (Choe & Cho, 1994). Hence, wet transfer of proteins from the gel to blotting supports is performed. Wet transfer is the process of electro-transferring polypeptides from a polyacrylamide gel to blotting supports by an electric field to enable immunoblotting techniques to be carried out (Lin & Kasamatsu, 1983).
Blotting supports include nylon, polyvinylidene difluoride (PVDF), and nitrocellulose membranes (Renart, et al., 1996). Nitrocellulose membranes are the first and most common membranes used to hold proteins through hydrophobic interactions, though electrostatic forces may also be involved (Choe & Cho, 1994).
Although most proteins bind spontaneously to nitrocellulose (Choe & Cho, 1994), they do not easily diffuse off the polyacrylamide gel (Rena, et al., 1996). Hence, an electric field which strengthens the binding of proteins to the nitrocellulose membrane (Choe & Cho, 1994) is utilized to aid the transfer process (Renart, et al., 1996).
Methanol is an essential component of the transfer buffer. It enhances hydrophobic interactions and thus, increases the transfer efficiency and affinity of proteins to the nitrocellulose membrane (Choe & Cho, 1994). This is especially so for smaller proteins which are less effectively transferred (Lin & Kasamatsu, 1983). In addition, methanol also removes excess SDS present (Choe & Cho, 1994). However, methanol may cause high molecular weight proteins to precipitate in the gel (Choe & Cho, 1994), and thus not entirely transferred to the membrane.
Before carrying out Western blot, the membrane on which the proteins are now on needs to be blocked. This is to prevent unspecific antibody-binding to any contaminant proteins which can result in high background (Renart, et al., 1996). Common blocking agents include solutions of detergents such as Tween-20, purified proteins such as serum albumin, and complex protein mixtures such as non-fat dry milk (Choe & Cho, 1994).
220.127.116.11 Western Blot
Western blot is the most prevalent immunochemical method (Choe, 1994) used to detect specific proteins with the use of antibodies (Renart, et al., 1996).
Incubation with suitable dilutions of primary antibodies is done to allow them to bind to their specific proteins for subsequent detection by labeled secondary antibodies (Renart, et al., 1996). Dilutions of these antibodies vary and are determined experimentally (Renart, et al., 1996). Typically, dilutions range from 1:100 to 1:1000 for polyclonal antibodies, 1:10 to 1:100 for hybridoma supernatants which are essentially monoclonal antibodies, and 1:1000 or more for ascites fluid-containing monoclonal antibodies (Renart, et al., 1996). Thus, it is crucial to optimise the dilution(s) of primary antibodies in order for efficient detection of proteins. In addition, the specificity and affinity of primary antibodies determine incubation times, which commonly ranges from 1 hour at room temperature to overnight at 4oC (Renart, et al., 1996).
Enzymes can directly be coupled to primary antibodies or indirectly linked to secondary antibodies (Choe, 1994). Primary antibodies bind to their specific proteins to form immunocomplexes which are detected by secondary antibodies. Secondary antibodies, like primary antibodies, have to be diluted to an optimum concentration before addition for maximum detection efficiency (Renart, et al., 1996). Most commercial products uses dilutions of 1:1000 to 1:3000 (Renart, et al., 1996). Usually, these added secondary antibodies are directly or indirectly conjugated to enzymes (Renart, et al., 1996). Direct binding involves bifunctional cross-linking agents such as glutaraldehyde and periodate (Renart, et al., 1996), which have mild reactivity at physiological pH and long half-lives in aqueous media (Choe, 1994). Indirect binding uses an avidin (streptavidin)-biotin bridge between secondary antibodies and enzymes (Choe, 1994 & Renart, et al., 1996). This increases sensitivity through amplification, but may give false-positive results due to biotin-binding proteins on the nitrocellulose membrane (Renart, et al., 1996). These enzymes in turn, acts on added substrates to produce colour or fluorescence that can be detected.
Horseradish peroxidase (HRP) and bacterial alkaline phosphatase are commonly employed enzymes (Renart, et al., 1996). In this experiment, horseradish peroxidase was used. Peroxidases are enzymes that oxidize substrates in the presence of hydrogen peroxide (Renart, et al., 1996). HRP does so by removing hydrogen atoms from substrates which are essentially electron donors, and reduces hydrogen peroxide into water molecules. When substrates are oxidised, they emit light or become coloured precipitates which can then be readily detected. The rates at which colour develops or light emits vary and are dependent on factors like the amount of proteins and detecting antibodies present, and the blocking solution used (Pierce Biotechnology, 2010). Figure 2.1 illustrates how HRP catalyses the oxidation of substrates using TMB as an example.
Figure 2.1. Oxidation of TMB by HRP. TMB loses two electrons in a two-step reaction and becomes a blue product of wavelength 450nm (Diamandis, et al., 1996).
HRP oxidize substrates such as chromogenic 4-chloronapththol (4CN), 3,3'-diaminobenzidine (DAB) and 3,3',5,5'-tetramethylbenidine (TMB) and luminescent 5-amino-2,3-dihydro-1,4-phtalazine dione (Luminol) (Renart, et al., 1996). Of which, TMB is most commonly used due to its less toxic properties and high sensitivity (Diamandis, et al., 1996). It is also known for its long term stability and availability (Diamandis, et al., 1996).