1. ELECTROPHORES: A Qualitativ Analysis of Protein and DNA
Dr. Gatot Ciptadi
FAKULTAS PETERNAKAN - UB

2. Now that we can cut DNA with restriction enzymes…
we can cut up DNA from different people… or different organisms… and compare it
why?
forensics
medical diagnostics
paternity
evolutionary relationships
and more…

3. Uses: Paternity
Who’s the father? –
Mom F1 F2
child
DNA  +

4. Comparing cut up DNA How do we compare DNA fragments?
separate fragments by size
How do we separate DNA fragments?
run it through a gelatin
gel electrophoresis
How does a gel work?

5. Gel electrophoresis – + DNA moves in an electrical field…
so how does that help you compare DNA fragments?
size of DNA fragment affects how far it travels
small pieces travel farther
large pieces travel slower & lag behind
DNA        – +
“swimming through Jello”

6. Gel Electrophoresis - + DNA & restriction enzyme wells gel
longer fragments
power
source
gel
shorter fragments +
completed gel

7. Running a gel 1 2 3 fragments of DNA separate out based on size
cut DNA with restriction enzymes 1 2 3
Stain DNA
ethidium bromide binds to DNA
fluoresces under UV light

8. Basic knowledge
Electrophoresis is a separations technique that is based on the the mobility of ions in an electric field.
Positively charged ions migrate towards a negative electrode and negatively-charged ions migrate toward a positive electrode.
For safety reasons one electrode is usually at ground and the other is biased positively or negatively.
Ions have different migration rates depending on their total charge, size, and shape, and can therefore be separated.
Gel electrophoresis is a technique used for the separation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein molecules using an electric current applied to a gel matrix.

9. What is Gel Electrophoresis?
Electro = flow of electricity, phoresis, from the Greek = to carry across
A gel is a colloid, a suspension of tiny particles in a medium, occurring in a solid form, like gelatin
Gel electrophoresis refers to the separation of charged particles located in a gel when an electric current is applied
Charged particles can include DNA, amino acids, peptides, etc

10. A gel being run Instrumentations) Power supplay
Comb
Power supplay
Kotak (electrophoresis chamber) tempat buffer
Buffer sebagai media elektrik
Sekat tempat gel
Gel sebagai medium yang akan dilewati sampel (filter molekul sampel)
Sampel yang diamati (DNA, protein)
Positive electrode
Agarose block
DNA loaded in
wells in the agarose
Buffer
Black background
To make loading wells easier

11. Electrophoresis Equipment
Power supply
Cover
Gel tank
Electrical leads 
Casting tray
Gel combs

12. How does it work?
DNA is an organic acid, and is negatively charged (remember, DNA for Negative)
When the DNA is exposed to an electrical field, the particles migrate toward the positive electrode
Smaller pieces of DNA can travel further in a given time than larger pieces

13. Loading the Gel
DNA
buffer  
wells   
Carefully place the pipette tip over a well and gently expel the sample. The sample should sink into the well. Be careful not to puncture the gel with the pipette tip.
Anode
(positive)
Add enough electrophoresis buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer.
Cathode
(negative)

14. + - Power • DNA is negatively charged.
• When placed in an electrical field, DNA will migrate toward the positive
pole (anode). H  O2
• An agarose gel is used to slow the movement of DNA and separate by size. + -
Power
Scanning Electron Micrograph of Agarose Gel (1×1 µm) 
• Polymerized agarose is porous,
allowing for the movement of DNA

15. + - How fast will the DNA migrate? Power
strength of the electrical field, buffer, density of agarose gel…
Size of the DNA!
*Small DNA move faster than large DNA
…gel electrophoresis separates DNA according to size
DNA + -
Power
small
large
Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.

16. Type of Gel
1. SDS-PAGE
SDS-PAGE stands for sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and is useful for molecular weight analysis of proteins.
SDS is a detergent that dissociates and unfolds oligomeric proteins into its subunits.
The SDS binds to the polypeptides to form complexes with fairly constant charge to mass ratios.
The electrophoretic migration rate through a gel is therefore determined only by the size of the complexes. Molecular weights are determined by simultaneously running marker proteins of known molecular weight.

17. 2. Agarose Gels
While acrylamide gels have become the standard for protein analysis, they are less suitable for extremely high molecular weight nucleic acids (above 200,000 daltons).
In order to properly separate these large molecules, the acrylamide concentration needs to be reduced to a level where it remains liquid.

18. Contoh hasil
Kiri: hasil dengan SDS-PAGE; kanan: hasil dengan agarose gel elektrophoresis

19. PAGE Apparatus (purity and MW)
Alat dan teknik Separasi (Protein)
PAGE Apparatus (purity and MW)

20. Slab Gel Electrophoresis (SGE)

22. Teknik dan hasil Separasi ELECTROFORESIS

23. Agarose gel electrophoresis
separation of DNA (RNA) fragment according to the molecular weight in % agarose gels
for DNA with a MW< 100 polyacrylamide gels may be used

24. DNA Ladder Standard DNA migration DNA migration -  12,000 bp  5,000
 100
 200
 300
 1,650
 1,000
 500
 850
 650
 400
 12,000 bp
 5,000
 2,000
DNA
migration
DNA
migration
Note: bromophenol blue migrates at approximately the same rate as a 300 bp DNA molecule
bromophenol blue +
Inclusion of a DNA ladder (DNAs of know sizes) on the gel makes it easy to determine the sizes of unknown DNAs.

25. Visualization of electrophoresis
300 bp
500 bp
1000 bp
2000 bp
A gel as seen under UV light - some samples had 2 fragments
of DNA, while others had none or one

26. Ethidium Bromide requires an ultraviolet light source to visualize

27. Visualizing the DNA (ethidium bromide)
 100
 200
 300
 1,650
 1,000
 500
 850
 650
 400
5,000 bp
 2,000
DNA ladder 
PCR Product
wells
Primer dimers
Samples # 1, 4, 6 & 7 were positive for Wolbachia DNA

28. Biotechnology
Gel Electrophoresis