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Home , Blot (biology), Western blot

SDS-PAGE and Western

Molecular Basis of Evolution

• Homology – high level of DNA and 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

• The number of proteins expressed by a species contributes more to its complexity than does the number of genes. – , antigens, , 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 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 – • 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 – 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 () – 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 – – 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

• - “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 .

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 () or Dry milk is used for blocking

22 Western Blot • Detection – Primary antibody specific to the protein of interest is added to the membrane • - 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 .

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 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 Of deletion mutants in the Hst1 protein

25 Western Blot

• Western Blot applications • Disease diagnosis – Used by medical technicians to confirm positive results for diseases such as HIV or . • 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.

26 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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