1. Gel Electrophoresis

2. Gel Electrophoresis Technique used for separation of
Deoxyribonucleic acid (DNA)
Ribonucleic acid (RNA)
Protein molecules
Using an electric current applied to a gel matrix

3. Gel Electrophoresis
Uses electricity to separate charged molec. on a gel slab
Separation based on size, shape and charge
Gel:
Powdered agarose (carb. derived from seaweed)
Dissolve in boiling buffer soln.
Most common agarose is polyacrylamide (PAGE)
Gel solidify and placed in get box and covered with buffer soln.

4. Gel Electrophoresis

5. Gel Electrophoresis

6. Separation
“Gel” refers to matrix used to contain, then separate target molecules
Gel is a crosslinked polymer whose composition and porosity is chosen based on specific weight and composition of target to be analyzed
"Electrophoresis" refers to electromotive force (EMF) that is used to move molecules through gel matrix
By placing molecules in wells in gel and applying an electric current, molecules will move through matrix at different rates
Usually determined by mass
Toward the positive anode if negatively charged
Toward the negative cathode if positively charged

7. Visualization After Electrophoresis Is Complete….
Molecules in gel can be stained to make them visible
Ethidium bromide
Silver Stain
Coomassie blue dye
Other methods may also be used to visualize separation of mixture's components on gel
If analyte molecules fluoresce under ultraviolet light, a photograph can be taken of gel under ultraviolet lighting conditions

8. Visualization After Electrophoresis Is Complete….
If several mixtures have initially been injected next to each other, they will run parallel in individual lanes
Depending on number of different molecules
Each lane shows separation of components from original mixture as one or more distinct bands
One band per component

9. Visualization After Electrophoresis Is Complete….
Incomplete separation of components can lead to overlapping bands, or to indistinguishable smears representing multiple unresolved components
Bands in different lanes that end up at same distance from top contain molecules that passed through gel with same speed
usually means they are approximately the same size
There are molecular weight size markers available that contain a mixture of molecules of known sizes
Marker run on one lane in gel parallel to unknown samples
Bands observed can be compared to those of unknown in to determine their size

10. Visualization After Electrophoresis Is Complete….
Agarose gel prepared for DNA analysis
The first lane contains a DNA ladder for sizing
Other four lanes show variously-sized DNA fragments that are present in some but not all of the samples

11. Applications Gel electrophoresis is used in:
Forensics
Molecular biology
Genetics
Microbiology
Biochemistry
Results can be analyzed quantitatively by visualizing gel with UV light and a gel imaging device

12. Nucleic acids Direction of migration
From negative to positive electrodes
Due to naturally-occurring negative charge carried by their sugar-phosphate backbone
Gel electrophoresis of large DNA or RNA is usually done by agarose gel electrophoresis

14. Proteins
Proteins, unlike nucleic acids, can have varying charges and complex shapes
They may not migrate into gel at similar rates, or at all, when placing a negative to positive EMF on sample
Proteins are usually denatured in presence of a detergent such as sodium dodecyl sulfate (SDS)
SDS coats proteins with a negative charge
Amount of SDS bound is relative to size of protein (usually 1.4g SDS per gram of protein), so that resulting denatured proteins have an overall negative charge
Proteins are usually analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

15. Proteins
SDS-PAGE - The indicated proteins are present in different concentrations in the two samples

16. DNA Electrophoresis
Analytical technique used to separate DNA fragments by size
An electric field forces fragments to migrate through a gel
DNA molecules normally migrate from negative to positive potential due to net negative charge of phosphate backbone of DNA chain
At scale of the length of DNA molecules
Gel looks much like a random, intricate network
Longer molecules migrate more slowly because they are more easily 'trapped' in network
After separation is completed
Fractions of DNA fragments of different length are often visualizing a fluorescent dye specific for DNA
Ex: ethidium bromide

17. DNA electrophoresis
Fragment size is usually reported in "nucleotides", "base pairs" or "kb"
Fragment size determination is typically done by comparison to commercially available DNA ladders containing linear DNA fragments of known length
Types of gel most commonly used for DNA electrophoresis are:
Agarose (for relatively long DNA molecules)
Polyacrylamide (for high resolution of short DNA molecules)
Gels have conventionally been run in a "slab" format
DNA strand is cut into smaller fragments using DNA endonuclease
Samples of DNA solution (DNA sample and buffer) are placed in wells of gel and allowed to run for some time
The less the voltage of electophoresis, the longer time for DNA sample to run through gel, and this results in a more accurate separation

18. Migration of DNA Fragments in Agarose
Fragments of linear DNA migrate through agarose gels with a mobility inversely proportional to log10 of their molecular weight
In other words, if you plot distance from well that DNA fragments have migrated against log10 of either their MW or number of base pairs, a roughly straight line will appear
Circular DNA migrate in agarose distinctly differently from linear DNAs of same mass
Uncut plasmids will appear to migrate more rapidly than same plasmid when linearized
Image shows an ethidium-stained gel with uncut plasmid in left lane and same plasmid linearized at a single site in right lane

19. Migration of DNA Fragments in Agarose
Several additional factors have important effects on mobility of DNA fragments in agarose gels:
1. Agarose Concentration
2. Voltage
3. Electrophoresis Buffer
4. Effects of Ethidium Bromide

20. 1. Agarose Concentration
Using different concentrations gels resolve different sizes of DNA fragments
Higher concentrations of agarose for small DNAs
Low agarose concentrations allow resolution of larger DNAs
Image shows migration of a set of DNA fragments in 3 concentrations of agarose
All in same gel tray and electrophoresed at same voltage and for identical times
Notice:
Larger fragments are much better resolved in 0.7% gel
While small fragments separated best in 1.5% agarose
1000 bp fragment is indicated in each lane

21. 2. Voltage
As voltage applied to a gel is increased, larger fragments migrate proportionally faster that small fragments
So, for best resolution of fragments larger than about 2 kb is attained by applying no more than 5 volts per cm to gel
The cm value is distance between two electrodes, not the length of the gel

22. 3. Electrophoresis Buffer
Several different buffers have been recommended for electrophoresis of DNA
Most commonly used for DNA are:
TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA)
DNA fragments will migrate at somewhat different rates in these two buffers due to differences in ionic strength
Buffers not only establish a pH, but provide ions to support conductivity
If you mistakenly use water instead of buffer, there will be essentially no migration of DNA in the gel
If you use concentrated buffer (e.g. a 10X stock solution), enough heat may be generated in gel to melt it.

23. 4. Effects of Ethidium Bromide
Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels
It can be incorporated into agarose gels, or added to samples of DNA before loading to enable visualization of fragments within gel
Binding of ethidium bromide to DNA alters its mass and rigidity, and therefore its mobility

24. Agarose & Polyacrylamide
Most commonly used support matrices
Provide a means of separating molecules by size (porous gels)
Porous gel may act as a sieve by retarding, or in some cases completely obstructing, movement of large macromolecules while allowing smaller molecules to migrate freely
Dilute agarose gels are generally more rigid and easy to handle than polyacrylamide of same concentration
Agarose is used to separate larger macromolecules such as nucleic acids, large proteins and protein complexes
Polyacrylamide, which is easy to handle and to make at higher concentrations, is used to separate most proteins and small oligonucleotides that require a small gel pore size for retardation

25. Agarose & Polyacrylamide
Handout

26. Separation of Proteins and Nucleic Acids
Proteins are amphoteric compounds
Their net charge therefore is determined by pH of medium in which they are suspended
In a solution with a pH above its isoelectric point, a protein has a net negative charge and migrates towards anode in an electrical field
Below its isoelectric point, protein is positively charged and migrates towards cathode
Nucleic acids remain negative at any pH used for electrophoresis
Carry a fixed negative charge per unit length of molecule, provided by the PO43- group of each nucleotide of nucleic acid
Electrophoretic separation of nucleic acids is strictly according to size

27. SDS- PAGE OF PROTEINS
Separation of Proteins under Denaturing conditions
SDS is an anionic detergent which denatures proteins by "wrapping around" polypeptide backbone
SDS confers a negative charge to polypeptide in proportion to its length
In denaturing SDS-PAGE separations migration is determined not by intrinsic electrical charge of polypeptide, but by molecular weight

28. Determination of Molecular Weight
Done by:
SDS-PAGE of proteins
PAGE or agarose gel electrophoresis of nucleic acids - of known molecular weight along with protein or nucleic acid to be characterized
A linear relationship exists between logarithm of molecular weight of an SDS-denatured polypeptide, or native nucleic acid, and its Rf
Rf is calculated:
As ratio of distance migrated by molecule to that migrated by a marker dye-front
A simple way of determining relative molecular weight by electrophoresis (Mr):
Plot a standard curve of distance migrated vs. log10MW for known samples
Read off logMr of sample after measuring distance migrated on same gel

29. Agarose Polysaccharide extracted from seaweed
Typically used at concentrations of 0.5 to 2%
The higher agarose concentration the "stiffer" the gel
Agarose gels are extremely easy to prepare:
You simply mix agarose powder with buffer solution, melt it by heating, and pour gel. It is also non-toxic
Agarose gels have a large range of separation, but relatively low resolving power
By varying concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be separated using standard electrophoretic techniques

30. Polyacrylamide Cross-linked polymer of acrylamide
length of polymer chains is dictated by the concentration of acrylamide used
Typically between 3.5 and 20%
Polyacrylamide gels are more annoying to prepare than agarose gels
Because oxygen inhibits polymerization process, they must be poured between glass plates (or cylinders)
Acrylamide is a potent neurotoxin and should be handled with care!
Polyacrylamide is considered to be non-toxic, but polyacrylamide gels should also be handled with gloves due to possible presence of free acrylamide

31. Polyacrylamide
Polyacrylamide gels have a rather small range of separation, but very high resolving power
Case of DNA, polyacrylamide is used for separating fragments of less than about 500 bp
Under appropriate conditions, fragments of DNA differing is length by a single base pair are easily resolved
In contrast to agarose, polyacrylamide gels are used extensively for separating and characterizing mixtures of proteins

32. Gel Electrophoresis

33. Gel Electrophoresis

34. Gel Electrophoresis Gel stains: Nucleic acids are colorless
Must be stained
DNA stains:
Ethidium bromide (EtBr)…orange when mixed with DNA under UV light
Methylene blue…dark blue…not as sensitive as EtBr viewed with white light

35. Gel Electrophoresis most common Sizing standard Only one DNA type
Plasmid restriction digestion
DNA sample from bacterial chromosome
RNA
Smears (thousands of different size molec in small concentration)
No nucleic acids
DNA so large will not load
Ex: eukaryotic genome

36. Proteins
Companies that produce protein products or study proteins must be able to:
Separate the protein of interest
Determine that amount of protein present.
Characteristics of proteins that make it possible to achieve either one of both points above:
Overall charge, size, shape, and solubility

37. Proteins
SDS-PAGE
Gel electrophoresis allows for the separation of proteins based on charge, size, and shape.
Polyacrylamide gel electrophoresis is utilized (PAGE).
Allows for better resolution
4-18% gels most commonly used
Higher concentration for smaller proteins
When protein size unknown gradient gels can be used.
Less concentrated at the top than the bottom

38. Proteins SDS-PAGE Use of sodium dodecyl sulfate (SDS)
Denatures proteins into polypeptide strands
Gives each polypeptide strand an overall negative charge
Proteins studied are strictly being separated by size

40. Proteins SDS-PAGE Visualization of proteins in gel
Coomassie Blue
Milligram amounts of protein.
Silver stain
Microgram amounts of protein.
Size of unknown bands can be determined from comparison to protein molecular weight standard

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