SDS-PAGE and Western Blot. Molecular Basis of Evolution
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1 1 SDS-PAGE and Western Blot Molecular Basis of Evolution Homology high level of DNA and protein sequence similarity due to common ancestry. Evidence Genomes of related organisms are very similar. Even if the DNA sequences of two related organisms is very different, the amino acid sequences may be the same or very similar.
2 2 Diversity of Proteins The number of proteins expressed by a species contributes more to its complexity than does the number of genes. Enzymes, antigens, antibodies, messenger proteins, structural proteins Many of these proteins are conserved among many organisms. Some of these proteins have evolved over time while others have remained relatively unchanged. Muscle Proteins Complex proteins such as proteins involved with muscles have remained relatively unchanged over time We will use muscle proteins to determine the evolutionary relatedness among a group of organisms.
3 3 Skeletal Muscle Voluntary muscle. Examples biceps, triceps. Contraction moves the bones in your body Innervated by motor nerves. Skeletal Muscle Muscle fibers appear striated. Multi nucleated cells. Muscle Muscle Fibers Myofibrils Sarcomere Mitochondria Provide ATP to muscle.
4 4 Skeletal Muscle Thin filaments Two strands of actin and one strand of tropomyosin (a regulatory protein) coiled around each other. Thick filaments An array of myosin molecules Repeating subunits of myofibrils make up a sarcomere Gives skeletal muscle its striated appearance.
5 Skeletal Muscle Z lines the borders of the sarcomere. Thin filaments attached to the Z line I band Contains only thin filaments A band Corresponds to the length of the thick filaments. H zone Center of the A band Contains only thin filaments 5
6 6 Skeletal Muscle The length of the sarcomere is reduced when the muscle contracts The distance between the Z lines becomes shorter The A bands do not change in length. The I bands shorten The H zone disappears. Skeletal Muscle Sliding filament model Neither the thin or thick filaments change in length when the muscle contracts. The degree of overlap of thick and thin filaments increases when the muscle contracts.
7 7
8 8 Skeletal Muscle Remember that there are other proteins besides actin and myosin which are also involved in muscle contraction which show more variability across different organisms! We will look at these other proteins to determine the evolutionary relatedness among species.
9 9 Evolution and Classification of Fishes Fish belong to the Phylum Chrodata Evolution and Classification of Fishes Class Chondrichthyes (cartilaginous fishes) Sharks and rays Cartilaginous skeleton Skin is thick and without scales. No swim bladder or lungs
10 Evolution and Classification of Fishes Class Osteichthyes (bony fish) Ray-finned fishes bass, trout, perch, tuna and herring Lobe-finned fishes known from the fossil record Lungfishes inhabit stagnant ponds and swamps Evolution and Classification of Fishes Class Agnatha (jawless fish) Lampreys and hagfishes Eel-like jawless fishes with parasitic and scavenging lifestyles 10
11 11 PAGE polyacrylamide gel electrophoresis Electrophoresis the migration of charged molecules in an electric field toward an electrode with the opposite charge.
12 12 After protein expression SDS-PAGE can answer the following questions What proteins are being expressed in my sample? What are the molecular weights of the proteins? What differences are there in the proteins from different sources? How pure is my protein of interest? How much protein do I have? One Dalton the mass of a hydrogen atom. Protein molecular weights are measured in Kd (kilodaltons) Very small as compared to most nucleotide chains which are millions of Kd Proteins are made up of amino acids which can have a positive, negative or neutral charge. This is a problem when trying to separate proteins using electrophoresis.
13 13 Denaturation disrupting the structure of a protein. Secondary, teritary and quartenary structure are disrupted by heat, SDS and BME (beta mercaptoethanol) SDS (sodium dodecyl sulfate) Ionic/denaturing detergent Associates with proteins All of the proteins are negatively charged. Allows proteins to be separated by size rather than charge. The proteins are treated with SDS and denatured. Denaturing creates linear amino acid chains. SDS coats the amino acids so that the entire protein has a negative charge. The proteins can now be separated by mass only.
14 14
15 15 Running Buffer Glycine allows for discontinuous electrophoresis. SDS keeps the samples denatured and of a constant charge. Tris maintains a proper ph Sample buffer 0.1% bromophenol blue tracking dye 300mM Beta-mercaptoethanol reducing agent disrupts the disulfide bonds in the proteins, 20% glycerol high density solution 2% running buffer (SDS/glycine/Tris) Polyacrylamide gel matrix Able to separate smaller molecules than agarose gels since the pore size is smaller. Used to separate protein molecules and very small nucleotide molecules (sequencing of nucleotides using a sequencing gel) The higher the concentration of polyacrylamide the smaller the molecule that can be separated. 5% polyacrylamide Kd 18% polyacrylamide 5 30 Kd
16 16 To cast a gel, a reaction initiator ammonium persulfate (APS) and a catalyst tetramethylethlenediamine (TEMED) are added to a polyacrylamide solution. Powdered or liquid unpolymerized acrylamide are neurotoxins!! We will use pre-cast polyacrylamide gels which are safe to use.
17 17 Discontinuous gel electrophoresis The gel consists of a stacking layer and a resolving layer. Stacking Gel Makes sure the protein samples enter the resolving gel at the same time. Large pore matrix - 4% polyacrylamide gel. Cl- (leading ions) have a greater mobility than the proteins Glycine (trailing ions) in stacking gel have a slower mobility than the proteins. Faster ions produce zone of low conductivity between themselves and the protein. Faster migrating proteins slowed down by the ions
18 18 Separating/resolving gel Smaller pore size 5% to 20% polyacrylamide gel. No ion gradient Separates the proteins based on size not charge. The gels are stained with Coomassie protein stain so that the bands appear blue.
19 19 Protein standards are used to determine protein sizes. SDS-PAGE _G_pC8U
20 20 Western Blot Antibody - protein used by the immune system to identify and neutralize foreign objects like viruses and bacteria. Each antibody recognizes a specific antigen Antibodies are proteins. Produced in animals such as mouse or goat Introduce the antigen of interest and then harvesting the antibodies produced against that antibody. Western Blot Antigen A substance that stimulates an immune response, especially the production of antibodies Can be proteins or polysaccharides
21 21 We will look for differences in protein banding patterns of various fish. Differences indicate how closely or distantly related the fish are. A thick band indicates an abundance of protein. We cannot determine the identity of a protein through SDS-PAGE. The identity of a protein can be determined through Western Blot Western Blot Proteins transferred to a membrane and then stained. Immunoblotting or Western Blotting is used to identify specific proteins (also known as antigens) by using antibodies which bind to the specific protein. The name is a play on words based on the Sourthern Blot developed by Edwin Southern.
22 22 Western Blot Transfer (Blotting) After SDS-PAGE the proteins are transferred to a nitrocellulose membrane. The negatively charged proteins move towards the anode. Blocking All proteins bind to the membrane. The protein of interest as well as other proteins Since the membrane binds all proteins the antibody must be blocked from binding to the membrane. The antibody must only bind to the protein of interest. 1% BSA (bovine serum albumin) or Dry milk is used for blocking Western Blot
23 23 Detection Western Blot Primary antibody specific to the protein of interest is added to the membrane Monoclonal antibody - one antibody which recognizes only one antigen. Polyclonal antibody - different antibodies which recognize the same antigen. Detection continued The membrane is rinsed to remove any non-bound primary antibody A secondary antibody is added which binds to the primary antibody The secondary antibody is bound to an enzyme. Western Blot
24 24 Western Blotting Detection continued A substrate is added that reacts with the enzyme to create a product that can be visualized. The product produced is either color or light. If there is product from the enzyme then the protein is present.
25 25 Western Blotting Results The results of this experiment tell us whether a specific protein is there or not. It doesn t tell us anything about the functionality of the protein. Western Blot An example of a Western blot used to detect expression Of deletion mutants in the Hst1 protein
26 26 Western Blot Western Blot applications Disease diagnosis Used by medical technicians to confirm positive results for diseases such as HIV or Lyme disease. Agricultural applications Detect and quantify proteins produced by genetically modified crops. Detect disease. Western Blot Biochemical and Biomedical Applications Detect post translational modifications of proteins glycosylation (look for change in m.w.) cleavage of a protein (antibodies which detect cleaved protein) phosphorylation (antibodies which bind only when a protein has been phosphorylated) Detect how new drugs affect protein structure. Detect changes in protein expression over time. Isolate a protein of interest and determine amino acid sequence by mass spectrohotometry.
27 27 2-D Electrophoresis Use the same techniques as SDS- PAGE. Proteins are first separated according to charge and then separated according to molecular weight.
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