Electrophoresis and blotting Ann Babtie Tech talk, 8th July 2014 8 July 2014 Electrophoresis and blotting 1 / 9 Introduction Gel electrophoresis separate mixtures of DNA, RNA or proteins by size (and charge) used to identify, quantify, purify and/or analyse biomolecules molecules migrate through a polymer gel under influence of electric field Blotting separate biomolecules by gel electrophoresis, then transfer to a nitrocellulose or PVDF membrane for further analysis Southern (DNA), northern (RNA) and western (protein) blots 8 July 2014 Electrophoresis and blotting 2 / 9 Agarose gel electrophoresis (nucleic acids) DNA/RNA migrate from - to + electrode due to phosphates Detected by fluorescent intercalating dyes (e.g. EtBr) Migration rate/distance depends on size (and conformation) _ migration direction + (Schaerli et al, NAR 2010) 8 July 2014 Electrophoresis and blotting 3 / 9 PAGE: Polyacrylamide gel electrophoresis (proteins) Usually use denaturing conditions (SDS-PAGE) | detergent (SDS) and reducing agents (e.g. DTT or βME) Proteins migrate from - to + due to bound (negative) SDS (BioRad manuals) 8 July 2014 Electrophoresis and blotting 4 / 9 PAGE variants (proteins) `Native' gels (non-denaturing conditions) are used to separate proteins on basis of natural charge and conformation 2D gel electrophoresis separates proteins by two different properties (usually isoelectric focusing in one direction, then SDS-PAGE in perpendicular direction) 8 July 2014 Electrophoresis and blotting 5 / 9 Blotting Southern (DNA), northern (RNA) and western (protein) blotting After electrophoresis, transfer biomolecules from gel to membrane for further immunological or biochemical analyses Usually electrophoretic transfer but other methods available (e.g. capillary action) 8 July 2014 Electrophoresis and blotting 6 / 9 Southern (DNA) & northern (RNA) blots Identify specific DNA/RNA by (nucleic acid) probe hybridisation Probe labelled to allow detection: e.g. radioactive or fluorescent label; or link to enzyme with chromogenic or chemiluminescent substrates Example applications: probe genomic DNA with cDNA probe (Southern), detect gene expression (northern) 8 July 2014 Electrophoresis and blotting 7 / 9 Western blotting (or immunoblotting) Identify specific protein(s) using primary antibody Secondary antibody detection: fluorescent colorimetric chemiluminescent radioactive 8 July 2014 Electrophoresis and blotting 8 / 9 Extra notes sometimes use polyacrylamide gels for (small) nucleic acids, or agarose for (large) proteins huge range of different sample preparation, gel types and detection/visualisation options other types of blotting (e.g. eastern, northwestern, southwestern, far-eastern, ...) much less common and in some cases ill-defined 8 July 2014 Electrophoresis and blotting 9 / 9.