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Unit 2

GEL ELECTROPHORESIS: Gel electrophoresis is a method for separation and analysis of biological

macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge.

Principles: Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially

proteins and nucleic acids - that differ in size, charge or conformation. As such, it is one of the most widely-used

techniques in biochemistry and molecular biology.

When charged molecules are placed in an electric field, they migrate toward either the positive or negative

pole according to their charge. Negative charged molecules migrate toward the anode and positive towards cathode.

Proteins and nucleic acids are electrophoresed within a matrix or "gel". Most commonly, the gel is cast in the shape

of a thin slab, with wells for loading the sample. The gel is immersed within an electrophoresis buffer that provides

ions to carry a current and some type of buffer to maintain the pH at a relatively constant value. The gel itself is

composed of agarose or polyacrylamide, each of which has attributes suitable to particular tasks.

Types of GE:

1. Agarose or horizontal GE: Agarose is a polysaccharide extracted from seaweed. It is a linear polymer, made

up of the repeating monomeric unit of agarobiose. Agarobiose is a disaccharide made up of D-galactose and 3,6-

anhydro-L-galactopyranose. It is typically used at concentrations of 0.5 to 2%. The higher the 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 the gel. It is also non-toxic. Agarose gels have a large range of

separation, but relatively low resolving power. By varying the concentration of agarose, fragments of DNA from

about 200 to 50,000 bp can be separated using standard electrophoretic techniques. For DNA.

a. Materials required:

 Agarose

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 1X TAE buffer (40 mM Tris, 20mM Acetic acid, 1 mM EDTA, pH 8.0)

 DNA sample(s)

 DNA loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 40% sucrose/glycerol in water)

 DNA ladder i.e molecular wt marker

 Ethidium bromide staining solution (0.01g of ethidium bromide in 1 L of MilliQ H2O)

 Gel tray, gel caster, Gel comb

 Horizontal electrophoresis unit

 Power supply

 UV transilluminator

B. Procedure for DNAseparation through agarose GE

a. Gel Casting/Prepration: The gel is prepared by dissolving the agarose powder in an appropriate buffer, such as

TAE or TBE, to be used in electrophoresis. The agarose is dispersed in the buffer before heating it to near-boiling

point. The melted agarose is allowed to cool sufficiently before pouring the solution into a caster. A comb is placed

in the caster to create wells for loading sample, and the gel should be completely set before use. The concentration

of gel affects the resolution of DNA separation. For a standard agarose gel electrophoresis, a 1% gives good

separation or resolution of large 5–10kb DNA fragments, while 2% gel gives good resolution for small 0.2–1kb

fragments. 1% gels are common for many applications. High percentage gels are often brittle and may not set

evenly, while low percentage gels (01.-0.2%) are fragile and not easy to handle. Low-melting-point (LMP) agarose

gels are also more fragile than normal agarose gel. The ethidium bromide may be added to the agarose solution

before it solidifies, or the DNA gel may be stained later after electrophoresis process.

b. DNA samples Loading: Once the gel has set/solidify, the comb is removed, leaving wells where DNA samples

can be loaded. Loading buffer is mixed with the DNA sample before the mixture is loaded into the wells. The

loading buffer contains a dense compound, which may be glycerol, sucrose, or Ficoll that raises the density of the

sample so that the DNA sample may sink to the bottom of the well. If the DNA sample contains residual ethanol

after its preparation, it may float out of the well. Loading buffer also include colored dyes such

as xylenecyanol and bromophenol blueused to monitor progress of electrophoresis. DNA samples are loaded using

a pipette.

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c. Process of electrophoresis: Agarose gel slab in electrophoresis tank with bands of dyes indicating progress of the

electrophoresis after current supply. The DNA moves towards anode. Agarose gel electrophoresis is most

commonly done horizontally in a submarine mode whereby the slab gel is completely submerged in buffer during

electrophoresis. It is also possible, but less common, to perform the electrophoresis vertically, as well as

horizontally with the gel raised on agarose legs using the appropriate apparatus. The buffer used in the gel is the

same as the running buffer in the electrophoresis tank, which is why electrophoresis in the submarine mode is

possible with agarose gel.

For optimal resolution of DNA > 2kb in size in standard gel electrophoresis, 5 to 8 V/cm is recommended

(the distance in cm refers to the distance between electrodes, therefore this recommended voltage would be 5 to 8

multiply by the distance between the electrodes in cm). Voltage may also be limited by the fact that it heats the gel

and may cause the gel to melt if it is run at high voltage for a prolonged period, especially if the gel used is LMP

agarose gel. Too high a voltage may also reduce resolution, as well as causing band streaking for large DNA

molecules. Too low a voltage may lead to broadening of band for small DNA due to dispersion and diffusion.

d. Staining and Visualization of DNA: Since DNA is not visible in natural light, the progress of the electrophoresis

is monitored using colored dyes. Xylene cyanol (light blue color) comigrates large DNA fragments, while

Bromophenol blue (dark blue) comigrates with the smaller fragments. Less commonly-used dyes include Cresol

Red and Orange G which migrate ahead of bromophenol blue. A DNA marker is also run together for the

estimation of the molecular weight of the DNA fragments.

The gel is visualized in UV illumination, and DNA stained with ethidium bromide appears as glowing

orange bands.DNA as well as RNA are normally visualized by staining with ethidium bromide, which intercalates

into the major grooves of the DNA and fluoresces under UV light. Other methods of staining are available,

examples are SYBR Green, GelRed, methylene blue, brilliant cresyl blue, Nile blue sulphate, and crystal

violet. SYBR Green, GelRed and other similar commercial products are sold as safer alternatives to ethidium

bromide as it has been shown to bemutagenic in Ames test, although the carcinogenicity of ethidium bromide has

not actually been established. SYBR Green requires the use of a blue-light transilluminator. DNA stained with

crystal violet can be viewed under natural light without the use of a UV transillumintor which is an advantage,

however it may not produce a strong band.

When stained with ethidium bromide, the gel is viewed with an ultraviolet (UV) transilluminator. Standard

transilluminators use wavelengths of 302/312-nm (UV-B), however exposure of DNA to UV radiations for as little

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as 45 seconds can produce damages to DNA and affect subsequent procedures. Exposure of the DNA to UV

radiations therefore should be limited. Using higher wavelength of 365 nm (UV-A range) causes less damage to the

DNA but also produces much weaker fluorescence with ethidium bromide. Where multiple wavelengths can be

selected in the transillumintor, the shorter wavelength would be used to capture images, while the longer

wavelength should be used when it is necessary to work on the gel for any extended period of time.The

transilluminator apparatus may also contain image capture devices (known as gel documentation system), such as a

digital or polaroid camera, that allow an image of the gel to be taken or printed. The separated DNA bands are

often used for further procedures, and a DNA band may be cut out of the gel as a slice, dissolved and purified. The

gels may also be used for blotting techniques.

Application of AGE:

 Estimation of the size of DNA molecules following restriction enzyme digestion, e.g. in restriction

mapping of cloned DNA.

 Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting

 Separation of restricted genomic DNA prior to Southern transfer or of RNA prior to Northern transfer.

Results

2.i. SDS-PAGE (for>10 kb protein): Polyacrylamide is a cross-linked polymer of acrylamide. The length of the

polymer chains is dictated by the concentration of acrylamide used, which is typically between 3.5 and 20%.

Polyacrylamide gels are significantly more annoying to prepare than agarose gels. Because oxygen inhibits the

polymerization process, they must be poured between glass plates (or cylinders). Acrylamide is a potent neurotoxin

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and should be handled with care. Wear disposable gloves when handling solutions of acrylamide, and a mask when

weighing out powder. Polyacrylamide is considered to be non-toxic, but polyacrylamide gels should also be

handled with gloves due to the possible presence of free acrylamide. Polyacrylamide gels have a rather small range

of separation, but very high resolving power and generally used for protein seperation. In the case of small

fragment DNA, polyacrylamide is also used for separation but less than about 500 bp. In contrast to agarose,

polyacrylamide gels are used extensively for separating and characterizing mixtures of proteins. SDS-PAGE was

performed as per following protocol using a mini gel system with 12% acrylamide at 12/24 mA current. Wide

range mol wt marker was used to identify and assess the molecular weights of protein bands observed in the gel.

PAGE is performed as Laemmli method (1970).

A. Isolation/extraction of proteins

1. Heat shock treatment: Take 107 cells/ml and pellet them. Wash the pellet with Phosphate buffer saline

(PBS) twice/thrice and freeze thaw them 4-5 times in liquid nitrogen and at 37C in water respectively.

Then keep at -20C for downstreaming.

2. By ultrasonication: Take pellet of 107cells/ml. Sonication (> 20 kHz) of suspended pellet in 1x PBS is

done for two periods of 1-3 min each in ice at medium amplitude for complete lysis of cells.

3. By Chemical Method: Take pellet of 107cells/ml. Add 500 µl of cell lysis buffer. Lysis Buffer: NET+

Triton X100 (100 mM NaCl, 1 mM EDTA, 20 mM Tris)

Put it for 5-10 min for lysis of cell. Spin cells at speed for 10 minutes at 2-4oC to pellet nuclei. Take

supernatant for protein estimation. Or use Lysozyme for bacterial cell wall rupture and digest PM due to

osmotic effect of suspending in buffer.

4. Mechanical: By using blender, homogenizer, French press.

B. Materials required:

Acrylamide stock:

Proper ratio of acrylamide and bis-acrylamide were dissolved in 60 ml distilled water and was made up to 100 ml.

The stock solution was stored at 40C. A ratio between acrylamide and bisacrylamide of 19:1 is suitable for the

separation of small peptides, whereas a ratio of 29:1 is commonly used for the separation of “normal sized”

proteins. High molecular weight proteins are best separated using a 37:1 mix ratio.

1.5 M Tris-HCl (pH 8.8): For running gel

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18.15 g of Tris was dissolved in 85 ml distilled water. The pH was adjusted to 8.8 with 1 N HCl. The solution was

made up to 100 ml and stored at 4oC.

0.5 M Tris-HCl (pH 6.8): For stacking gel

6.0 g of Tris was dissolved in 70 ml distilled water. The pH was adjusted to 6.8 with 1N HCl. The solution was

made up to 100 ml. and stored at 4oC.

SDS (10%)

10 g of SDS was dissolve in 100 ml distilled water and stored at room temperature.

Ammonium per Sulphate (APS) 10%

2 g of APS was dissolved in 20 ml distilled water, aliquoted and stored at –20oC.

Tris-glycine electrophoresis buffer (pH 8.3)

(25 mM Tris base, 250 mM Glycine, 0.1% SDS)

Tris - 3.0 g

Glycine - 18.4 g

SDS – 1.0 g

The above reagents were dissolved in 990 ml distilled water. The pH was adjusted at 8.3, stored at RT.

Sample loading dye/buffer

(0.125 M Tris-Cl, pH 6.8, 4% SDS, 20% Glycerol 10% 2- mercaptoethanol, Bromophenol blue)

2.5 ml - 4 X Tris-Cl (Solution No. 3)

4.0 ml - 10 % SDS (Solution no. 4)

2.0 ml - Glycerol

0.2 ml - - mercaptoethanol

0.2 mg - Bromophenol blue

The above solutions were dissolved in 10 ml distilled water, aliquoted into 1 ml and stored at 4-8 0C.

Coomassie Brilliant blue stain:

0.25 g of Coomassie Brilliant Blue was dissolved in 90 ml of methanol: H2O (1:1). Finally 10 ml of glacial acetic

acid was added followed by filtration with filter paper.

Destaining solution:

90 ml of 1:1 (v/v) Methanol: distilled water and 10 ml of acetic acid was dissolved.

C. Procedure for SDS-PAGE:

1. Glass plates were assembled according to the manufacturer’s instructions.

2. 12 % acrylamide solution was prepared for the separating gel.

3. Acrylamide solution was poured into the gap between the glass plates and left sufficient space for the

stacking gel. Stacking and running gel is prepared as per above chart and according to % choice.

4. The acrylamide solution was overlayed with isobutanol and the gel was placed in a vertical position at

room temperature.

5. After polymerization was completed (30 min), the overlay was poured off and the top of the gel was

washed several times with deionised water to remove any unpolymerized acrylamide.

6. 3 % Stacking gel was prepared using the appropriate volume of acrylamide solution.

7. The stacking gel solution was poured directly onto the surface of the polymerized resolving gel.

8. A clean Teflon comb was inserted immediately and carefully into the stacking gel solution, to avoid

trapping air bubbles. The gel was placed in a vertical position at room temperature.

9. After polymerization was completed (30 min), the Teflon comb was removed carefully. Using a squirt

bottle, the wells were washed immediately with deionised water to remove any unpolymerized acrylamide.

10.The gel was mounted in the electrophoresis apparatus.

11.Tris-glycine electrophoresis buffer was added to the top and bottom reservoirs. Any bubbles that had

become trapped at the bottom of the gel between the glass plates were removed with a bent hypodermic

needle attached to a syringe.

2. Sample preparation: Protein molecular weight marker and protein sample were prepared by heating them to

100 0C in equal volume of 2x SDS sample-loading buffer to denature the protein.

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3. Electrophoretic separation of proteins in SDS-PAGE:

 Each well was charged with 25 µl of prepared sample having 300µg protein with a Hamilton microliter

syringe. The syringe was washed with buffer after the loading of each sample. Equal volume of 1x SDS

sample loading buffer was poured into the unused wells also.

 Electrophoretic apparatus was attached to an electric power supply at 12 mA current.

 After the dye front had moved into the separating gel (30 min), current was increased to 24 mA and run

the gel until the bromophenol blue reached the bottom of the resolving gel (about 2-4 hours). Then the

power supply was turned off.

 The glass plates were removed from the electrophoresis apparatus and placed them on a paper towel.

Using a spatula, the plates were pried apart and cutting a corner from the bottom of the gel that is closest

to the leftmost well marked the gel orientation.

 The gel was now be fixed and stained with Coomassie Brilliant Blue.

STAINING OF SDS-PAGE:

1. Coomassie Brilliant blue (CBB) staining of SDS-PAGE gels:

SDS-PAGE gels were simultaneously fixed with methanol: glacial acetic acid and stained with CBB powder as:

1. The gel was immersed in at least 5 volumes of staining solution.

2. Placedit on a slowly rotating platform for a minimum of 2-4 hours at room temperature.

Destaining of CBB gel: To remove the stain, the gel was distained by soaking it in the methanol/acetic acid

solution without the dye on a slowly rocking platform for 4-8 hours. Destaining was performed in destaining

solution [1:1 (v/v) Methanol: TDW and 10 ml of acetic acid)]. After distaining, gels were stored indefinitely in

water containing 20% glycerol until photographed or scanned.

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2. Silver staining of SDS-PAGE gels:

It is used when more sensitive method for detection is needed, as classical CBB staining. CBB can usually detect a

100 ug protein band, Silver staining increases the sensitivity typically 50 times. Silver staining was introduced by

Kerenyi and Gallyas as a sensitive procedure to detect trace amounts of proteins in gels. The technique has been

extended to the study of protein macromolecules because it is more sensitive for protein detection. Many variables

can influence the colour intensity and every protein has its own staining characteristics. The colour produced by the

early silver stains ranged between light yellow and an orange-red. Steps for silver staining

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3. Other/special types of GE

A. 2D Gel electrophoresis (2DGE):

2-DGE, first described in 1975 is a powerful method for separating proteins in biological fluids or cell lysates, and

a good visualization tool for protein expression where the proteome profiles of control and disease samples can be

compared to differentiate physiological states. 2-DGE separates proteins in a two step approach according to their

pI (Iso electric point is the pH at which the net charge of the protein is zero), and molecular mass (MW) in a gel

matrix under denaturing conditions. Separation in the first dimension of analytical 2-DGE is known as Isoelectric

focusing which is a separation technique which separates peptides according to their isoelectric point, or how acidic

and basic their residues are. It is carried out using immobilized pH gradient (IPG) strips (Earlier manual gel was

prepared with career ampholyte for 1D), whereas the second dimension separation is performed using SDS-PAGE.

IPG Types
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1. Broad Range: 3-10, 4-11, 2. Narrow Range: 4-7, 6-9, 3. Micro range: 4-5, 6-7. (Strip length: 7, 11, 18, 24 cm)

Vertical Mini Gel System size (l x b x h)=15x18x15; Vertical Midi Gel = 16x20x15; Vertical Maxi Gel

size=25x20x27. Mini Gel Size (cm) 8x7, Midi gel size: 16x14, Maxi gel: 16x20

IPG strips

Using immobilized pH gradient (IPG) gels for first dimension IEF eliminates the problems associated with carrier

ampholytes. The immobilized pH gradient (IPG) gel is formed by casting polyacrylamide gels using acrylamido

buffers (acrylamide derivatives containing charged groups) on a plastic backing. Since the pH gradient is cast into

the gel, the gradient is more stable and reproducible. After the IPG gel is polymerized, the gel is washed, dried, and

cut into narrow strips (IPG Strips).The pH gradient on the IPG strip can be linear or nonlinear (NL). The non-linear

gradient is usually expanded from pH 4-7 resulting in a sigmoidal pH gradient. The non-linear IPG strips are useful

for analyzing samples containing many proteins with pI in the range of 4-7, which is typical for most crude lysates

from all species. Advantages of IPG strips

 More stable and reproducible pH gradients

 Reduced cathodic drift

 Higher mechanical strength of IPG because the strips are cast on a plastic backing minimizing gel breakage

 Higher protein loading capacity due to the sample loading method

Advantage: 2-DGE provides information about the intact proteins. Furthermore, protein isoforms with post-

translational modifications changing their net charge are often well separated and can be independently quantified.

Disadvantages include discrimination against certain classes of proteins such as hydrophobic, very basic and small

(<10 kDa) proteins. Furthermore, the limited loading capacity of 2-DGE often necessitates enrichment and pre-

fractionation steps in order to detect low-abundant proteins.

1D (IEF): Also known as Isoelectric focusing (IEF) or electrofocusing, is a technique for separating

different molecules by differences in their isoelectric point (pI). pI is the pH at which a particular moleculecarries

no net electrical charge i.e. the negative and positive charges are equal. A protein that is in a pH region below

its (pI) will be positively charged so will migrate towards cathode (negatively charged electrode) & vice versa.

Step

 Isolation and quantification of proteins (1mg/ml protein).

 Dissolve isolated protein in rehydration buffer (Urea, Tris, DTT, T-X-100. Tributyl phosphine).

 Put rehydrated protein in cassette and also IPG strip in cassette over rehydrated protein. Incubate for ½-2 hr
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 After that add mineral oil in cassette keep it room temperature overnight (12-16 hrs).

Next day take out IPG strip from cassette and put strip on tissue paper vertically to remove the mineral oil. Now

transfer IPG strip into IEF cell and run in under the following conditions: (i) conditioning step for 20 min at 250 V,

(ii) liner voltage ramping for 2 hr and 30 min at 10,000 V, (iii) for the final focusing step 10,000 V constant until

40 kVh, and the current did not exceed 50µA/strip.

2D (SDS-PAGE): After IEF, the strip was equilibrated in solution A (0.375 M Tris, pH 8.8 containing 6 M urea,

2% SDS, 20% glycerol, 2% w/v DTT) and B (solution A without DTT, but with 2.5% w/v iodoacetamide) for 20

min at room temperature and inserted (90 degree) on to a 12% 2-D SDS-PAGE gel (20 x 22 cm) with wide range

prestained protein molecular weight marker. It was used to identify and assess the molecular weights of protein

bands observed in the gel. The gel was sealed with 1% agarose and SDS-PAGE was performed according to the

Laemmli method (1970) as described earlier. After that gel was stained with CBB/Silver and stained gels were

scanned and images were acquired. Protein spots of interest were excised manually or by spot picking robotic

system from the gels. The spots were transferred to microcentrifuge tubes, labeled properly and stored at -80C for

its identification by mass spectrometry.

B. Pulse- Field Gel Electrophoresis (PFGE):

Normal and standard GE techniques for separation of DNA molecules provided huge advantages for molecular

biology research. However, it was unable to separate very large molecules of DNA effectively (> 10 kb). This

technique became known as pulsed-field gel electrophoresis because in PFGE an electric field is provided

periodically with changes direction. The development of PFGE expanded the range of resolution for DNA
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fragments by as much as two orders of magnitude. The procedure for this technique is relatively similar to

performing a standard gel electrophoresis except that instead of constantly running the voltage in one direction, the

voltage is periodically switched among three directions; one that runs through the central axis of the gel and two

that run at an angle of 60 degrees either side. Electric field alternates 120 degree every 90 sec for 18-24 hrs at 14
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C. The pulse times are equal for each direction resulting in a net forward migration of the DNA. For extremely

large bands (up to around 2Mb), increases the pulse time for each direction over the course of a number of hours

sake.This procedure takes longer than normal gel electrophoresis due to the size of the fragments being resolved

and the fact that the DNA does not move in a straight line through the gel. All large fragments of DNA run at the

same rate, and appear in a gel as a single large diffuse band. Thus separation of very large DNA pieces using PFGE

is made possible. PFGE may be used for genotyping or genetic fingerprinting. It is commonly considered a gold

standard in epidemiological studies of pathogenic organisms.

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C. Capillary gel electrophoresis (CGE):

CE is electrophoresis performed in a capillary tube. It is the most efficient separation technique available for the

analysis of both large and small molecules. The transformation of conventional electrophoresis to modern CE was

spurred by the production of inexpensive narrow-bore capillaries for gas chromatography (GC) and the

development of highly sensitive on-line detection methods for high performance liquid chromatography

(HPLC).The basic instrumental set-up, consists of a high voltage power supply (0 to 30 kV), a fused silica (SiO2)

capillary, two buffer reservoirs, two electrodes, and an on-column detector. Gel size is 0.1 mm in diameter and

capillary tube 50-80 cm in length. Sample injection is accomplished by temporarily replacing one of the buffer

reservoirs with asample vial. A specific amount of sample is introduced by controlling either the injection voltage

or the injection pressure. A detector is placed at the end of capillary for fluorescent detection. Capillary

electrophoresisis the most efficient separation technique available for the analysis of both large/ small molecules.

RESULT

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