Gel electrophoresis Tutorial - PowerPoint Presentation

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Gel electrophoresis

Tutoriallise schoonen14-12-’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 fieldMarker 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 gels1969 - Reliable MW determination of proteins using SDS1972 - 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 separationAdvantage: 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: SDSSDS-PAGENative 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

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Name

Structural formula

FunctionAcrylamidePolymerizes to form the gelTris pH 8.8BufferSDS (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

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SDS-PAGE -

Separation mechanism

Power on

Glycine moves towards anode, into stacking gel

Glycine ions become zwitterionic and slow downChlorine 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

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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 speed9

SDS-PAGE -

Visualisation

Coomassie Brilliant Blue

Anionic dye that binds proteins non-specifically

Silver stainingMore sensitive than Coomassie stainingCan 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 seaweedAgarose is thermally set, no polymerization reaction neededPercentage 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 modelDescribes 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 DNAGel concentration Higher concentration reduces migration speedElectric 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

MutagenicAlternatives: SYBR Green, SYBR Safe15

Agarose GE - Visualisation

Southern blotting

Detect specific DNA sequence in DNA sample

Method:

Run agarose gelTransfer 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

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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’ DNA17

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 science18

Moving boundary electrophoresis

Developed by Arna Tiselius

Electrophoresis

free in solution

Nobel prize in Chemistry in 1948The 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.19

MW determination proteins using SDS

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