Protein Immunological Analysis Techniques PDF

DIDACTIC UNIT 2: MAIN TECHNIQUES Chapter 3 | Protein immunological analysis techniques Professor: Mónica Díez, PhD  IMMUNOLOGIC TECHNIQUES FOR PROTEIN ANALYSIS ✓Western blotting /Immunoblotting ✓Immunofluorescence ✓Immunoprecipitation ✓Arrays...

DIDACTIC UNIT 2: MAIN TECHNIQUES Chapter 3 | Protein immunological analysis techniques Professor: Mónica Díez, PhD  IMMUNOLOGIC TECHNIQUES FOR PROTEIN ANALYSIS ✓Western blotting /Immunoblotting ✓Immunofluorescence ✓Immunoprecipitation ✓Arrays 2  PROTEIN ANALYSIS Gel Electrophoresis (transporte bajo la acción de un campo eléctrico) Electrophoresis is the method of separating charged molecules in an electric field. Electrophoretic separations are most often performed in a semisolid matrix, such as a gel, rather than in free solution because gels enhance the separation of macromolecules, like proteins and nucleic acids. HOW? Gels enhance separation in two ways: First, gels reduce convective mixing of samples. Second, the natural pores found in gels act as molecular sieves (tamiz), which act to separate molecules based on their molecular size. Thus, gel electrophoresis is the method of choice when separating macromolecules based on their molecular size. 3  PROTEIN ANALYSIS Gel Electrophoresis We use gels of very diverse size and nature Indispensable analytical tool, useful: To separate and compare protein mixtures To evaluate purity of a protein To estimate the quantity of a protein To detect proteolysis To detect protein modifications To estimate physical characteristics of a protein (MW, pI,…) … for almost everything… 4  PROTEIN ANALYSIS Polyacrilamide gel electrophoresis, PAGE Electroforesis en geles de poliacrilamida, PAGE - 1, One-dimensional Electrophoresis in denaturing conditions (SDS-PAGE) - 3, One-dimensional Electrophoresis in NON-denaturing conditions -2, Two-dimensional Electrophoresis 5  POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) (One-dimensional Electrophoresis in denaturing conditions (SDS-PAGE)) Employing gel and buffer discontinuities to produce sharp separation among sample components (improve resolution) - a low percentage stacking gel (gel concentrador), sample components are stacked into very thin, sharp zones prior to the final separation - a separating gel (gel separador) in which the sample is fractionated SDS-PAGE electrophoresis of proteins (often referred to as the Laemmli system) multiphasic systems 6 POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) - a low percentage stacking gel, sample components are stacked into very thin, sharp zones prior to the final separation - a separating gel in which the sample is fractionated At the beginning of the experiment, the buffer compositions are different in the stacking gel, the separation gel, and the tank. - The stacking gel contains a Tris-HCl buffer at pH 6.8. - The separation gel contains a higher concentration Tris-HCl buffer at pH 8.8. - The tanks contain Tris-glycine at pH 8.8. http://www.youtube.com/watch?v=IWZN_G_pC8U http://www.youtube.com/watch?v=EDi_n_0NiF4 7  POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) -The stacking gel contains Tris-HCl buffer at pH 6.8. -The separation gel contains Tris-HCl buffer at pH 8.8. -The tanks contain Tris-glycine at pH 8.8. Gly- Gly- - Gly- Gly- Gly Gly- Gly- Gly- Gly- Cl- Cl- Gly- Cl- Gly- Cl- - Cl Gly- Cl- At the start of multiphasic With the establishment of the Electrophoresis in the SDS-PAGE protein Kohlrausch boundary in the resolving gel is electrophoresis the anions stacking gel, the proteins are indistinguishable from are chloride (green) in the stacked into a thin layer ordinary homogeneous stacking and resolving gels between the leading chloride buffer systems. and glycine (orange) in the ions and the trailing glycine tanks. molecules. 8 POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) The pore size of a gel is determined by two factors, the total amount of acrylamide present (%T) (T = Total concentration of acrylamide and bisacrylamide monomer) and the amount of cross-linker (%C) (C = bisacrylamide concentration). Pore size decreases with increasing %T; with cross-linking, 5%C gives the smallest pore size. 9  POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) The principle by which all proteins get stacked in the concentrating gel is named isotacophoresis. Chloride (leading ion) displays higher mobility than proteins, and proteins higher mobility than Glycine (trailing ion). Application of electrical power leads to a low electrical field in the leading ion zone and to a high electrical field in the trailing ion zone. As a consequence, all proteins get stacked in a narrow zone. 10  POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) Fig.1 Before SDS: Protein (pink line) incubated with the denaturing detergent SDS showing negative and positive charges due to the charged R-groups in the protein. The large H's represent hydrophobic domains where nonpolar R-groups hide away from the polar water that surrounds the protein. After SDS: SDS molecules disrupt hydrophobic areas (H's) and coat proteins with many negative charges which overwhelm any positive charges the protein had due to positively charged R-groups. As a consequence, proteins get denatured by SDS (reduced to its primary structure- aminoacid sequence) and become linearized. 11 POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) 12 POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) The detergent binds to hydrophobic regions in a constant ratio of about 1.4 g of SDS per gram of protein. SDS-PAGE involves separating proteins based on their size alone. SDS (sodium dodecylsulfate) is a detergent that denatures proteins. It disrupts their hydrophobic areas and coats them with negative charges. By doing so, proteins will retain simply their primary structure and all will have equivalent charge to mass ratios. 13 POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) Acrylamide and bis-acrylamide Polyacrylamide gels are composed of chains of polymerized acrylamide that are crosslinked by a bifunctional agent such as N,N' -methylene- bis-acrylamide A stock solution containing 29% (w/v) acrylamide and 1% (w/v) N,N'-methylene-bis-acrylamide 14  POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) Acrylamide and bis-acrylamide Cross-links formed from bisacrylamide add rigidity and tensile strength to the gel and form pores through which the SDS-polypeptide complexes must pass. The size of these pores decreases as the bisacrylamide:acrylamide ratio increases, reaching a minimum when the ratio is ~ 1:20. Most SDS-polyacrylamide gels are cast with a molar ratio of bisacrylamide:acrylamide of 1:29, which allows to resolve polypeptides of MWs differing in as little as 3%. 15  POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) Acrylamide and Bis-acrylamide polymerize Catalizador Prepare the small ammount you need fresh everytime 16  POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) Acrilamida y Bis-acrilamida polymerize IMPORTANTE 17 POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) 18 POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) 19 POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) 20 POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) 21 POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) To determine the size or molecular weight of unknown proteins, a series of standards (proteins of known molecular weight) treated similarly is electrophoresed along with the unknown proteins in adjacent lanes of the gel. By measuring the distance each protein travels in mm from the origin (i.e. well), one can determine the relative mobility (Rf) of each protein. The relative mobility of a protein can be calculated using the following equation: 22  POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) http://www.youtube.com/watch?v=pjEHJI PRtlU&NR=1&feature=fvwp This value can then be plotted on semi-log paper to generate a calibration curve against which the molecular weight of unknown size proteins can be determined. 23  POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) QUIZ Protein Name Molecular weight (KDa) Actin 42 p53 (also known as tumor protein 53) 53 Leptin (a protein hormone that plays a key role in regulating energy intake and energy expenditure) 16 Heat Shock Protein (folding and unfolding chaperone, many members, ex. Hsp27, Hsp70…) 33, 60, 70, 90 Single-stranded DNA-binding protein (or SSB has 4 identical 19 kDa subunits) 76 Glucose-regulated protein (in the endoplasmic reticulum of the cell, 3 isoforms) 78 / 94 / 170 Protein C (regulating blood clotting, inflammation, cell death, has 2-chains: 21 and 42 KDa) 62 Thyroglobulin (dimeric protein of the thyroid gland) 660 Protein A (cell surface protein) 56 Fatty acid synthase (FAS is a multi-enzyme protein that catalyzes fatty acid synthesis) 272 24  POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) EQUIPMENT 25  POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) EQUIPMENT http://www.youtube.com/watch?v=EDi_n_0Ni F4&feature=related Abnova http://www.youtube.com/watch?feature=ends creen&NR=1&v=in4i78hJDZY 26  POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) What happens after electrophoresis? 1. Fix the proteins in the gel and stain them OR 2. Electrophoretic transfer to a membrane and then probe with antibodies- (Immunoblotting = Western-blot). 27  POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) What happens after electrophoresis? 1. Fix the proteins in the gel and stain them OR 2. Electrophoretic transfer to a membrane and then probe with antibodies- (Immunoblotting = Western-blot). 28  POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) Western-blot protein Southern-blot DNA Northern-blot RNA 29 What happens after electrophoresis? TAP7.13 TAP7.14 1. Stain the proteins in the gelTAP7.1 TAP7.3 TAP7.4 TAP7.2 TAP7.8 TAP7.10 M M Example: Coomassie staining 225 150 102 76 52 WB_28 38 38 31 31 NuPAGE Novex Bis-Tris Mini Gel (4-12%) Running Buffer MOPS 24 24 12 12  Example: Silver staining TAP8_Analysis 1- ASF1 6- ASF1 5- RAD53 We will talk with more detail about staining in 2D-gels chapter  POLYACRILAMIDE GEL ELECTROPHORESIS, DENATURING CONDITIONS (SDS-PAGE) What happens after electrophoresis? 1. Fix the proteins in the gel and stain them OR 2. Electrophoretic transfer to a membrane and then probe with antibodies- (Immunoblotting = Western-blot). 32  Western blot - IMMUNOBLOTTING Immunoblotting is used to identify and measure the size of macromolecular antigens (usually proteins) that react with a specific antibody. Proteins are first separated by electrophoresis with SDS-polyacrylamide gels and then transferred electrophoretically from the gel to a solid support, such as a nitrocellulose, polyvinylidene difluoride (PVDF), or cationic nylon membrane. Antigen-antibody complexes are finally located by chromogenic, radiographic, or chemiluminescent reactions. * 33 Real gels are transparent  Western blot - IMMUNOBLOTTING Western blots allow investigators to determine the molecular weight of a protein and to measure relative amounts of the protein present in different samples. Proteins are separated by gel electrophoresis, usually SDS-PAGE. Why? The proteins are transferred to a sheet of special blotting paper called nitrocellulose, polyvinylidene difluoride (PVDF), or cationic nylon. The proteins retain the same pattern of separation they had on the gel. 34  Western blot - IMMUNOBLOTTING The blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the membrane. An antibody is then added to the solution which is able to bind to its specific protein. The second antibody has an enzyme (e.g. alkaline phosphatase or horseradish Why? peroxidase) or dye attached to it which cannot be seen at this time. The location of the antibody is revealed by incubating it with a colorless substrate that the attached enzyme converts to a colored product that can be seen and photographed in chromogenic reactions. 35 Western blot - IMMUNOBLOTTING 36 Western blot - IMMUNOBLOTTING Semy-dry Transfer Wet 37  Western blot - IMMUNOBLOTTING EQUIPMENT Below, equipment wet transfer of proteins from gel to membrane 38  Western blot - IMMUNOBLOTTING TRANSFER WET SEMI-DRY (-) (+) 39 COOMASSIE Semydry vs wet transfer Below, the same samples were subjected to SDS-PAGE in identical 16%-polyacrylamide gels. Protein transfer to membranes was performed and then gels were Coomassie-stained. 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 WB_37 SEMYDRY TRANSFER WB_38 WET TRANSFER Needs to be improved  Western blot - IMMUNOBLOTTING TRANSFER, different membranes Three types of membranes are used for immunoblotting: nitrocellulose, nylon, and polyvinylidene fluoride (PVDF). Different proteins may bind with different efficiencies to these membranes, and particular antigenic epitope(s) may be better preserved in one case than another. It is therefore worthwhile to test the efficiency with which the antigen of interest can be detected using different types of membranes, using antibodies. 41  Western blot - IMMUNOBLOTTING TRANSFER 1. Nitrocellulose (pore size 0.45 µm) remains a standard membrane used for immunoblotting, although membranes with a smaller pore size (0.22 µm or 0.1 µm) are recommended for immunoblotting of small proteins of MW