Tutorial lise schoonen 14-12-’15 Gel electrophoresis Tutorial lise schoonen 14-12-’15

What is gel electrophoresis? Method for separation and analysis of macromolecules DNA, RNA, proteins Separation based on size and/or charge Electric field Marker can be used to determine size of sample

History 1931 - First report of electrophoresis as a separation technique 1937 - Report on the ‘Tiselius apparatus’ for moving boundary electrophoresis 1955 - Introduction of starch gels 1959 - Introduction of acrylamide gels 1969 - Reliable MW determination of proteins using SDS 1972 - Agarose gel electrophoresis with ethidium bromide stain

Polyacrylamide gel electrophoresis (PAGE) Mostly used for protein separation (5-2000 kDa) Chemical polymerization Percentage acrylamide determined pore size, thus separation Advantage: high resolving power, also for small DNA fragments (5-500 bp’s) Disadvantage: small range of separation

PAGE gel methods Denaturing gels Native gels Native structure of macromolecule is disrupted, mobility depends on linear length Nucleid acids: urea | Proteins: SDS SDS-PAGE Native gels Analysis macromolecules in their folded state, size and shape influence mobility BN-PAGE: Coomassie Blue provides necessary charges CN-PAGE: No additional charges introduced to proteins QPNC-PAGE: Preparative variant

SDS-PAGE - Compounds involved Name Structural formula Function Acrylamide Polymerizes to form the gel Tris pH 8.8 Buffer SDS (sodium dodecyl sulfate) Anionic detergent to linearize proteins and to impart a negative charge to linearized proteins, leading to a constant mass to charge ratio for every protein APS (ammonium persulfate) Persulfate free radicals convert acrylamide monomers to free radicals which react with unactivated monomers to begin the polymerization chain reaction TEMED (tetramethylethylenediamine) Accelerates the rate of formation of free radicals from persulfate

SDS-PAGE - Polymerisation TEMED accelerates formation of free radicals from persulfate Persulfate radicals convert acrylamide monomers into radicals Acrylamide radicals react with unactivated monomers and bis-acrylamide

SDS-PAGE - Separation mechanism Power on Glycine moves towards anode, into stacking gel Glycine ions become zwitterionic and slow down Chlorine ions form ion front ahead of glycine towards anode Proteins reside between glycine and chlorine ions and become concentrated between the two fronts at high electric field When the running gel is reached, the pH increases, which causes deprotonation of the glycine ions and a high increase in their velocity The higher acrylamide concentration slows down the proteins according to size Laemmli buffer system

SDS-PAGE - Separation Separation depends on: Size Small proteins move faster Gel concentration Higher concentration reduces migration speed Electric field Higher voltage increases migration speed

SDS-PAGE - Visualisation Coomassie Brilliant Blue Anionic dye that binds proteins non-specifically Silver staining More sensitive than Coomassie staining Can also be used to visualize nucleic acids and polysaccharides Western blot Antibody-based detection Very sensitive

SDS-PAGE - Application Analysis of proteins in blood serum Two classes of proteins: serum albumin and globulin

Agarose gel electrophoresis Mostly used for nucleic acid separation (200-50.000 bp’s) Material: natural polysaccharide polymers extracted from seaweed Agarose is thermally set, no polymerization reaction needed Percentage agarose determines separation Advantages: large range of separation, easy sample recovery by gel extraction Disadvantage: relatively low resolving power

Agarose GE - Separation mechanism Ogston model Describes behaviour of DNA smaller than gel pores Molecules move through pores large enough to accommodate their passage. Movement of large molecules is impeded by collisions with gel matrix. Reptation model Describes behavious of DNA larger than gel pores DNA crawls through the matrix in a “snake-like” fashion

Agarose GE - Separation Separation depends on: Size Small fragments move faster Conformation Supercoiled DNA moves faster than relaxed DNA Ethidium bromide concentration Can change the charge and conformation of DNA Gel concentration Higher concentration reduces migration speed Electric field Higher voltage increases migration speed

Agarose GE - Visualisation Ethidium bromide Intercalates into the major grooves of DNA Fluoresces under UV light Mutagenic Alternatives: SYBR Green, SYBR Safe

Agarose GE - Visualisation Southern blotting Detect specific DNA sequence in DNA sample Method: Run agarose gel Transfer DNA to nitrocellulose membrane Expose to hybridization probe, including a radioactive label Wash away excess probe Visualize on X-ray film by autoradiography Northern blotting Similar to Southern blotting Used for RNA detection

Agarose GE - Application Paternity test DNA fingerprinting Restriction enzymes are used to cut DNA into pieces Pattern of the child should be a combination of the parents’ DNA

Conclusions Gel electrophoresis is a versatile technique for the analysis of proteins and nucleic acids Different types of gels Different visualization techniques It is applied in fields ranging from clinical chemistry to forensic science

Moving boundary electrophoresis Developed by Arna Tiselius Electrophoresis free in solution Nobel prize in Chemistry in 1948 The apparatus includes a U-shaped cell filled with buffer solution and electrodes immersed at its ends. The sample applied could be any mixture of charged components like a protein mixture. On applying voltage, the compounds will migrate to the anode or cathode depending on their charges. The change in the refractive index at the boundary of the separated compounds is detected using Schlieren optics at both ends of the solution in the cell.

MW determination proteins using SDS