BMS2043 - Electrophoresis 2024.pdf

BMS2043- ANALYTICAL AND CLINICAL BIOCHEMISTRY ELECTROPHORESIS DR PENNY LYMPANY [email protected] 28AY04 OBJECTIVES At the end of this session you will be able to describe the differences between different types of electrophoresis and outline examples of their uses WHAT TOPICS ARE WE GOING TO CO...

BMS2043- ANALYTICAL AND CLINICAL BIOCHEMISTRY ELECTROPHORESIS DR PENNY LYMPANY [email protected] 28AY04 OBJECTIVES At the end of this session you will be able to describe the differences between different types of electrophoresis and outline examples of their uses WHAT TOPICS ARE WE GOING TO COVER? Agarose electrophoresis SDS-PAGE Capillary Electrophoresis Western Blotting WHY? Assess DNA, RNA & protein purity Characterize proteins Isolate peptides, DNA & RNA WHAT IS ELECTROPHORESIS? Electrical field - caution – Ohm’s law! V/I = R V=voltage I=current R=resistance Mobility – speed of migration = mobility Medium – agarose, polyacrylamide, capillary – pore size, ionic strength Separation – smaller more highly charged molecules move faster This Photo by Unknown Author is licensed under CC BY-SA TYPES OF ELECTROPHORESIS Agarose gel – DNA Polyacrylamide gel – high resolution, proteins and smaller nucleic acid. Smaller pore size vs agarose – better separation of small molecules Capillary – narrow capillary with buffer – high resolution, fast separation APPLICATIONS DNA fingerprinting DNA sequencing Protein analysis Clinical diagnosis – serum proteins, biomarkers Pharmaceutical development – characterisation of biologics e.g. antibodies, vaccines AGAROSE GEL ELECTROPHORESIS DNA & RNA Agarose: 0.5%-2% - higher conc = smaller pore sizes Horizontal Native structure Migration towards positive electrode Loading dye: bromophenol blue, xylene cyanol Staining: Ethidium bromide, non-toxic fluorescent stains Size determination DNA or RNA extraction SOUTHERN BLOT This Photo by Unknown Author is licensed under CC BY-SA-NC SDS-PAGE Proteins SDS: sodium dodecyl sulphate PAGE: polyacrylamide gel electrophoresis 5%-20% polyacrylamide Vertical Denatured protein (SDS, β-mercaptoethanol, boiling) Discontinuous buffer system Migration towards positive electrode SDS-PAGE METHODOLOGY Boiling SDS DTT/betamercaptoethanol https://www.technologynetworks.com/analysis/articles/polyacrylamide-gelelectrophoresis-how-it-works-technique-variants-and-its-applications-359100 SDS-PAGE: POLYMERIZATION SDS-PAGE Loading dye: Bromophenol blue Staining: Coomassie blue, nontoxic fluorescent stains Size determination Protein extraction (proteomics) SDS-PAGE: DISCONTINUOUS BUFFER SYSTEM Stacking gel (pH 6.8) Resolving gel (pH 8.8) http://technologyinscience.blogspot.co.uk/2012/06/disontinous-sds-page-two-phase-sds-page.html#.Vyr_v4SDFHy OTHER TYPES OF GELS SDS-PAGE method of choice but denatured protein during process Non-denaturing gels/native gels – separation, identification of protein of interest by substrate Gradient gels – for more complex mixtures, separation of similar Mr values Isoelectric focussing gels – based on pI (iso-electric points) 2D PAGE gels PROTEIN PURIFICATION: SDS-PAGE purification of the RecA protein of Escherichia coli SDS-PAGE: DETERMINING THE MOLECULAR WEIGHT OF A PROTEIN electrophoretic mobility of a protein on an SDS polyacrylamide gel is related to its molecular weight, Mr IMMUNOBLOTTING/WESTERN BLOTTING Transfer of products to a solid stable membrane – nitrocellulose Further investigate products – antibodies PROTEINS: ANTIBODY DETECTION IMMUNOBLOTTING ISOELECTRIC FOCUSSING 2D ELECTROPHORESIS CAPILLARY ELECTROPHORESIS – DNA SEQUENCING DNA SEQUENCING – PAGE GEL ELECTROPHORESIS VS CAPILLARY SEQUENCING Source: Lehninger https://www.sigmaaldrich.com/GB/en/technicaldocuments/protocol/genomics/sequencing/sanger-sequencing CAPILLARY ELECTROPHORESIS TO SEPARATE METABOLITES MORE INFORMATION ? ANY QUESTIONS? We made it! This Photo by Unknown Author is licensed under CC BY-SA Please use the discussion board for general questions and queries