1. ELECTROPHORESIS

2. Applications
Gel electrophoresis is used in forensics, molecular biology, genetics, microbiology and biochemistry
usually performed for :
Analytical purposes - -
Purify molecules

3. Basic principles of electrophoresis
It is the process of moving charged biomolecules in solution by applying an electrical field across the mixture.
Biomolecules moved with a speed dependent on their charge, shape, and size
Electrophoresis is used:
for analysis and purification of very large molecules (proteins, nucleic acids)
for analysis of simpler charged molecules (sugars, amino acids, peptides, nucleotides, and simpler ions).

6. Paper gel electrophoresis
Historical significance; first media used for electrophoresis by pioneering investigators like Tiselius.
Used in clinical investigations of serum and other body fluids.
Poor conductivity
* Background staining
* Non-transparent
* Can be stored easily

7. Starch gel electrophoresis
* Introduced by Smithies (1955).
* Starch used as Supporting media.
2 forms –
α amylose (unbranched) &
amylopectin (branched) polymers.
* Mostly used for protein separation.
Cooking potato starch with buffer until a uniform consistency is achieved.
* Good for proteins.
Problems :
Degassing is required

8. Cellulose acetate electrophoresis
* Hydroxyl groups of cellulose (paper) converted to acetyl groups
* Non-toxic
* Transparent
* Readily be dissolved in various solvents thus allowing easy recovery separated components
* Widely used in clinical applications

10. Gel Electrophoresis (i) Simple agarose gel electrophoresis
(ii) Simple polyacrylamide gel electrophoresis (PAGE)
(iii) Restriction Fragment Length Polymorphism (RFLP) analysis and oligomer restriction.

11. Gel Electrophoresis
Gel electrophoresis separates DNA and RNA molecules according to size, shape.
Gel matrix is an inserted, jell-like porous material that support and allows macromolecules to move through. Agarose and polyacrylamide are two different gel matrices

12. Agarose – highly purified polysaccharide derived from agar (extracted from seeweed), long sugar polymers held together by hydrogen and hydrophobic bonds.
Acrylamide (CH2=CH-CO-NH2)
Polyacrylamide gel structure held together by covalent cross-links

13. When charged molecules are placed in an electric field, they migrate toward
either the positive (anode) or
negative (cathode) pole according to their charge.
Factors influenced electrophoresis mobility:
net charge of the molecule
size and shape
concentration of the molecule in solution

14. Simple agarose gel electrophoresis
polysaccharide agarose (poly d-galactose 3,6-anhydro-l galactose)
purified from marine algae
solid compound at room temperature…. powder

15. Agarose gels OH CH2OH O Use For the separation of
(1) large protein or protein complex
(2) polynucleotide 50-30,000 base-pairs
The pore size is determined by adjusting the concentration of agarose in a gel (normally in the rank of 0.4-4% OH O
CH2OH

16. Agarose matrix … molecular sieve
Average agarose gel … 0.8 and 4% agarose
1–2% .. separation of PCR amplimers … 100bp
Does not allow …separation of molecules less than approximately 15 nucleotides in length.
Higher the agarose concentration more dense polymer network

17. DNA separation by gel electrophoresis
large
moderate
small
After electr

18. Ladders….
Amplimer size determination…. molecular weight markers … added to an empty well

20. PAGE used for proteins and small pieces of DNA
Polyacrylamide gel electrophoresis (PAGE)
Electrophoresis in a polyacrylamide matrix separating or resolving molecules in a mixture under the influence of an applied electric field
PAGE used for proteins and small pieces of DNA
Polyacrylamide has smaller pore size- better resolution (2bp differences in DNA) and separates smaller molecules
Acylamide is synthesized (not natural like agarose)

21. A typical setup consists of a gel slab sandwiched between two glass plates, with the ends enclosed in upper and lower reservoirs of buffer
Samples to be run are loaded in wells at the top of the gel, in conjunction with tracking dye. An electrical voltage is applied between the upper and lower reservoirs, causing the samples to migrate down through the gel.

22. Once a gel has been 'run', it is treated to reveal the positions of the samples
Staining
Coomassie blue-sensitive to 0.1ug of protein
Silver- sensitive to 0.002ug of protein,
To calibrate the relative migrations of molecules of different size, a marker lane is often added, where samples of known size will migrate to reference positions

23. 4 components of PAGE gel
Acrylamide: Acrylamide forms long polymer chains
APS: Polymerization induced by APS (ammonium persulphate) which generates free radicals (charged oxygens)
TEMED: TEMED is a free radical stabilizer (N’N’N’N’-tetra methylene diamine)
Bisacrylamide: Bis acrylamide is a cross linking agent and links long polymers of acrylamide (N, N’-methylene bis acrylamide)
Pore size is determined by % acrylamide and the amount of cross linker
The copolymerization of acrylamide with methylene bisacrylamide produces a mesh-like network in three dimensions, consisting of acrylamide chains with interconnections formed from the methylenebisacrylamide

25. SDS PAGE
SDS is used in the gel mix.
Sodium dodecyl suphate - detergent
SDS is –ve charged (pH range 7 – 10)and binds to proteins, it denatures (unfolds) proteins and gives a net negative charge.
The negative charges proteins are strongly attracted toward an anode (positively-charged electrode) in an electric field.
Can be separated based on size alone

26. Native PAGE
No denaturing agents
Proteins separated based on size charge and shape.
Used when want to keep protein active to study conformation

28. 1. Over loaded
3. Frowning (run too hot)
4. Bent bands. Tearing
5 . too low conc.
2. Tearing

32. An ethidium-stained gel photographed under UV light
**Each band that you see is a collection of millions of
DNA molecules, all of the same length!!

36. Type of electrophoresis buffer (for Nucleic Acid)
Buffer used … affect the DNA separation
TAE (Tris-Acetate-EDTA) ..
Resolution of fragments larger than 4 kb
TBE (Tris-Borate-EDTA) ..
providing better resolution of 0.1–3 kb fragments
Reused a few times