...SDS-PAGE also called sodium dodecyl sulfate polyacrylamide gel electrophoresis is a technique used to separate proteins only by their size. SDS or sodium dodecyl sulfate is a detergent used to denature the proteins; they will unfold and will not have any secondary, tertiary or quaternary structure. Proteins will have a negative charge and they will move down the gel by their molecular weight. PAGE or polyacrylamide gel electrophoresis is a polymer of the neurotoxin acrylamide and a cross-linking agent called bis-acrylamide. Usually APS and TEMED are used as catalysts for the process of cross-linking. There are two different settings of running the polyacrylamide gel, denaturing gel and non-denaturing gel or native gel. Denaturing gel consists of using urea or SDS to denature or unfold proteins and subsequently separate them by their molecular weight. A non-denaturing or native gel is used primarily to analyze the tertiary and quaternary structure of proteins; therefore they do not need to be unfolded. SDS-PAGE is essential in protein analysis in different fields such as forensic, biochemistry, biotechnology, molecular biology and genetics. The SDS-PAGE gel has to be poured in two parts. The first gel that is poured is the resolving gel. This type of gel consists of 3 to 30 % acrylamide thus possessing small pores and a pH of 8.8. The second gel is called the stacking gel and it is poured on top of the resolving gel. Stacking gel has 8 to 12 % acrylamide, therefore...
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...Introduction IgG is an antibody probe that has an affinity for binding specifically to the IgG protein antigen. The research goal was to identify how closely the IgG of the tested species are related. This was accomplished by examining how goat IgG, specific for goat protein, responded to the same protein from cows, pigs, rats, and humans, and it was determined which animals have the most similar antigen binding reactions. The variability in each animal’s IgG protein was revealed by the level of affinity that the anti-goat IgG probe had for the species tested. Since the variability in each animal’s proteins is due to their genetic coding sequence this study clarified which species have the most similar genetic code. Immunoglobulins are the antigen-recognition molecules of B cells and are used as the main effector function in adaptive immunity. (Janeways, 2001) Their are five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM and all five Ig classes are present in mammals and are produced from B lymphocytes as part of the immune response system. (Urich, 1994) IgG antibodies are large molecules, having a total molecular weight of 150kDa, composed of two heavy (H) peptide chains weighing approximately 50kDa and two light (L) peptide chains approximately 25kDa. (Janeways, 2001). The region of Light and Heavy chains connected by a disulfide bond makes up the Fragment antigen binding (Fab) structure, while the remaining Heavy chain region is referred to as the...
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...Researchers have found that the plant protein inhibits H1N1, H3N2 and H5N1 subtypes. Of the medicinal plants studied, the Momordica Charantia plant has been reported to contain many antiviral properties (Pongthanapisith, Ikuta, Puthavathana, and Leelamanit 1). The seed of the M. Charantia was purified of the protein using Fast Protein Liquid Chromatography. The proteins are separated using Sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE). The technique involves placing the protein mixture on gel containing an immobilized pH gradient. The gel slows the passage of proteins and acts as a molecular filter. Samples are loaded into the SDS-polyacrylamide gel and the electric field is applied. The gel slows the passage of proteins and acts as a molecular filter separating different protein molecules according to their size. Smaller proteins move faster than larger proteins and are found near the bottom of the gel. The gel is treated with a coomassie blue stain that binds to the proteins and allows the researcher to visualize the protein bands. The SDS-PAGE method is better suited for separating smaller molecules like protein.(Nelson and Cox 93-94) Madin-Darby canine kidney cells were inoculated with Influenza virus and incubated. The number of infected cells was counted under a microscope. Proteins of the Momordica Charantia were added to the infected cells. After further incubation overnight, the infected cells were identified with nucleoprotein antibodies of...
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...The Brad assay * is one of the most common forms of measuring absorbance through a spectrometer * uses a a dye (coomassie Brilliant Blue G-250) that binds to proteins and is absorbed at a wavelength of 465- 598 nm * Fast, therefore can meaure a 96 plate in 5 minutes * Absorbance shift allows the measure the protein at the 598 nm wavelength by using the spectrometer, can be justified through the beer-lambert law * The standard curve can be used to estimate the concentration of an unknown protein in solution by * Taking in account their known absorbance levels * Use line equation from curve * The unknown protein conc. in a sample with a known absorbance level can be determined Bradford Vs. Lowry * Lowry * Uses alkaline conditions- reduction cu 2+ to Cu+ * Folin phenol reagent- yellow to blue * Peptide bonds for monocovalent copper and radicals of try and trp react with folin * Concentrations 0.1-1.0 ug/ml of protein * Can be interfered with easily * Slow 40-60 * Colour varies with proteins * Criticial timing with procedure * * Bradford * Based on absorbance shift from brilliant blue when bound to proteins * 465- 598 (when bound) * Acidic conditions * Binds tightly to protein, and inhibits binding sites (little inference) * Limitations * Strong basic buffers Detergents interfere with binding of the cosmic blue, causing...
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