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. 1 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. 2 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. 3 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. 4 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 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. 6 7 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. 8 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 9 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 SDS – PAGE Electrophoresis • PAGE – polyacrylamide gel electrophoresis • Electrophoresis – the migration of charged molecules in an electric field toward an electrode with the opposite charge. 11 SDS – PAGE Electrophoresis • 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? SDS – PAGE Electrophoresis • 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. 12 SDS – PAGE Electrophoresis • 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. SDS – PAGE Electrophoresis • 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. 13 14 SDS – PAGE Electrophoresis • 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) SDS – PAGE Electrophoresis • 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 100-300 Kd • 18% polyacrylamide 5–30 Kd 15 SDS – PAGE Electrophoresis • 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. 16 SDS – PAGE Electrophoresis • Discontinuous gel electrophoresis – The gel consists of a stacking layer and a resolving layer. SDS – PAGE Electrophoresis • 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 17 SDS – PAGE Electrophoresis • Separating/resolving gel – Smaller pore size 5% to 20% polyacrylamide gel. – No ion gradient – Separates the proteins based on size not charge. SDS – PAGE Electrophoresis • The gels are stained with Coomassie protein stain so that the bands appear blue. 18 SDS – PAGE Electrophoresis • Protein standards are used to determine protein sizes. SDS – PAGE Electrophoresis • SDS-PAGE • http://www.youtube.com/watch?v=IWZN _G_pC8U 19 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 20 SDS – PAGE Electrophoresis • 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. 21 Western Blot • Transfer (Blotting) – After SDS-PAGE the proteins are transferred to a nitrocellulose membrane. – The negatively charged proteins move towards the anode. Western Blot • 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 22 Western Blot • Detection – 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. Western Blot • 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. 23 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. 24 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 – http://www.youtube.com/watch?v=nqspgMGNsrU An example of a Western blot used to detect expression