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

2. 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

3. History
First report of electrophoresis as a separation technique
Report on the ‘Tiselius apparatus’ for moving boundary electrophoresis
Introduction of starch gels
Introduction of acrylamide gels
Reliable MW determination of proteins using SDS
Agarose gel electrophoresis with ethidium bromide stain

4. Polyacrylamide gel electrophoresis (PAGE)
Mostly used for protein separation ( 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

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

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

12. Agarose gel electrophoresis
Mostly used for nucleic acid separation ( 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

13. 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

14. 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

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

16. 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

17. 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

18. 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

19. 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.

20. MW determination proteins using SDS