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Gel electrophoresisTUTORIAL

LISE SCHOONEN

14-12-’15

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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

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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

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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

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PAGE gel methods• Denaturing 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

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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

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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

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SDS-PAGE - Separation mechanism

1. Power on

2. Glycine moves towards anode, into stacking gel

3. Glycine ions become zwitterionic and slow down

4. Chlorine ions form ion front ahead of glycine towards anode

5. Proteins reside between glycine and chlorine ions and become concentrated between the two fronts at high electric field

6. When the running gel is reached, the pH increases, which causes deprotonation of the glycine ions and a high increase in their velocity

7. The higher acrylamide concentration slows down the proteins according to size

Laemmli buffer system

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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

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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

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

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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

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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

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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

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

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Agarose GE - Visualisation• Southern blotting• Detect specific DNA sequence in DNA

sample• Method:

1. Run agarose gel2. Transfer DNA to nitrocellulose membrane3. Expose to hybridization probe, including a

radioactive label4. Wash away excess probe5. Visualize on X-ray film by autoradiography

• Northern blotting• Similar to Southern blotting• Used for RNA detection

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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

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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

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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.

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MW determination proteins using SDS