Experiment 3

Purification and electrophoresis

analysis of plasmid DNA & proteins
from Escherichia coli

Yeon Jae Chang

Electrophoresis

• A technique used to separate and purify macromolecules, especially

proteins and nucleic acids, that differ in size, charge, or conformation
by an electrical field
• Ex) Agarose gel electrophoresis,
SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
Agarose gel electrophoresis
Agarose
• Polysaccharide extracted from seaweed
• Formation of a porous matrix
• Fragile because of the formation of weak hydrogen / hydrophobic
bonds
• Typically used at concentrations of 0.5 to 2 % (w/v)
Agarose gel electrophoresis

• Standard lab procedure for separating DNA by size (length in base

pairs)
• Negatively charged DNA is moved toward positive electrode
• The longer the DNA, the slower it moves
• Non-toxic substance
Agarose gel electrophoresis
• DNA migrates toward the positive electrode (+) because of the net
negative charge in electric field
• The buffer serves as a conductor of electricity and controller of pH
• DNA loading dye has glycerol and dye
Glycerol: provides density to the sample to be sunk
Dye: visualization for tracking of sample migration
Agarose gel electrophoresis
Staining
• Ethidium bromide (EtBr)
DNA intercalator which is inserted into the spaces between the base
pairs
Used for detecting DNA/RNA
Sensitive and easy stain, but potent mutagen (harmful)
• RedSafeTM
Alternative to traditional EtBr staining
Less sensitive, but lower mutation than EtBr (Non-toxic, non-
mutagenic)
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE)
• Widely used to analyze the proteins in complex extracts
• Consisted of two gels (running gel and stacking gel)
I. Running (separating) gel : Proteins are resolved based on the MWs
II. Stacking gel : Proteins are concentrated prior to entering the running gel
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE)
• Polyacrylamide gel are formed by the chemical polymerization of
acrylamide and N,N’-methylenebisacrylamide (cross-linking agent)
∴ Have to control the size of the pores in the gel by adjusting the
concentration of acrylamide
• Polymerization occurs because of free oxygen radicals
• The higher concentration of acrylamide, the smaller pore size of the
gel
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE)
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE)
• 5X Sample buffer contains bromophenol blue (BPB), glycerol,
mercaptoethanol, and SDS
Bromophenol blue : Tracking dye
Glycerol : Settlement of protein samples into the bottom of wells
Mercaptoethanol : Reduction of disulfide bonds in the proteins (linearization)
SDS : Disruption of noncovalent bonds, denaturation, and imposition of
negative charge
∴ Sample buffer has to be mixed with protein samples before gel
electrophoresis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE)
• Folded proteins assume many different geometries and chemistries
→ Positively and negatively charged proteins migrate in different directions
→ Uniform geometry and charge/mass ratio by using sample buffer and
boiling
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE)
• In stacking gel (Chloride ion (Cl-) > Protein-SDS complex > Glycine)
→ Difference in chloride and glycine mobility sets up a voltage gradient
• In running gel, glycine has negative charge because of pH of running gel
→ Voltage difference is eliminated (Cl- > Glycine > Protein-SDS complex)

→ Proteins migrate at different rates because of the sieving

properties of the gel
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE)
Coomassie Brilliant Blue staining
• Anionic dye which binds to proteins
• Strong, noncovalent complex formation based on van der Waals
forces and electrostatic interactions
• More efficient binding with basic amino acids than acidic one
Discussion

1. Describe the chemical compounds and roles of 3 solutions in DNA

preparation (except washing and elution buffer)
2. How does the DNA and proteins are moved in electric field?
3. Describe the fluorescence mechanism of green fluorescent protein
and modified fluorescent proteins which we used
4. Describe the roles of each compound in 5X protein sample buffer
5. How could you control the pore size of SDS-PAGE gel?
6. Considering the experimental results, measure the size of fluorescent
proteins and plasmid DNA
7. Describe three bands which are marked in Syllabus and why this
phenomenon is appeared
Experimental procedure
SDS-PAGE Preparation

1. Prepare the running (12%) and stacking gel (5%) mixtures

2. Set up the gel cassette and pour water between the glasses for
checking leakage
3. Mix TEMED and running gel solution and pour the mixture
4. After solidification, pour stacking gel solution with TEMED and insert
the comb until solidification
Experimental procedure
SDS-PAGE analysis

1. Set up the SDS-PAGE gel in the cast facing the thin glass inside
2. Fill the tank with 1X TGS buffer
3. Load 5 μL protein standard and 10 μL protein samples in each well
4. Charge with 300V for 25 min
5. Check that it functions well by bubbles up
6. Take off the gel and put in the gel in staining solution
7. Change staining solution to water and boil using microwave
Experimental procedure
DNA preparation

1. Resuspend cells with 250 μL equilibration buffer containing RNAse

2. Add 250 μL cell lysis buffer and mix gently (inverting)
3. Add 350 μL neutralization buffer and mix gently (inverting)
4. Centrifuge at maximum rpm for 10 min
5. Pour the supernatant into the column and centrifuge for 30 sec. Then
discard the flow-through
6. Apply 700 μL washing buffer B and centrifuge for 30 sec. Discard the
flow-through. Then centrifuge the empty column for 1 min to remove
residual buffer
7. Transfer the column to a new 1.5 mL tube
8. Add 50 μL elution buffer. After 1 min, centrifuge for 1 min
Experimental procedure
Agarose gel preparation & electrophoresis

1. Mix 0.28 g agar powder and 40 mL 1X TAE buffer (0.7 %(w/v))

2. Dissolve the mixture by heat with microwave for 2 min
3. Add 5 μL RedSafeTM and mix well
4. Pour the solution to the mold and wait until it is solidified
5. Mix the purified DNA and 6X loading dye to 5:1
6. Set the gel to the cast and load 10 μL DNA and 5 μL DNA ladder
7. After 25 min, detect the DNA with UV illuminator
TA contact
장연재: [email protected] (Rm# 4116)

남윤호: [email protected]

정석영: [email protected]

홍유진: [email protected]

최로빈: [email protected]