Types and applications for gel electrophorasis

presented by: samah nasr elshaikh TABLE OF CONTENTS 01 History 02 Introduction 03 Diffreant types of gel 04 How to Prepare the gel ? 05 Applications of 01- History

First invention invented by Arne Tiselius, a Swedish scientist. •First electrophoresis, moving boundary/ Tiselius electrophoresis. •Then, he invented the new method “zone Arne Tiselius electrophoresis.

•Oliver Smithies invented starch gel electrophoresis.

Oliver Smithies 02 - introduction where dose the name “ Electrophoresis “ come from ?

where dose the name “ Electrophorasis “ come from ?

The suffix phoresis from the Greek = to carry across - means "migration" or "movement.“

The prefix electro = flow of electricity , tells us that we are using electricity to make molecules migrate.

Then Gel electrophoresis refers to the separation of charged particles located in a gel when an electric current is applied Gel electrophoresis is a technique used to separate DNA fragments (or other macromolecules, such as RNA and proteins) based on their size and charge.

Gel Electrophoresis involves running a current through a gel containing the molecules of interest. Based on their size and charge, the molecules will travel through the gel in different directions or at different speeds, allowing them to be separated from one another. What is a gel?

As the name suggests, gel electrophoresis involves a gel: a slab of Jello-like material. Gel is a cross linked polymer whose composition and porosity is chosen based on the specific weight of the target molecules .

Different types of gels which can be used are agar and agarose gel, starch, sephadex, polyacrylamide gels , cellulose acetate , which comes as dry, powdered flakes. When the powder is heated in a buffer (water with some salts in it) and allowed to cool, it will form a solid, slightly squishy gel. At the molecular level, the gel is a matrix of agarose molecules that are held together by hydrogen bonds and form tiny pores.

At one end, the gel has pocket-like indentations called wells, which are where the DNA samples will be placed .

How do DNA fragments move through the gel?

Once the gel is in the box, each of the DNA samples we want to examine is carefully transferred into one of the wells. One well is reserved for a DNA ladder, a standard reference that contains DNA fragments of known lengths. Commercial DNA ladders come in different size ranges, so we would want to pick one with good "coverage" of the size range of our expected fragments.

Next, the power to the gel box is turned on as one end of the gel has a positive charge and the other end has a negative charge. , and current begins to flow through the gel.

Because DNA and RNA are negatively charged molecules because of the phosphate groups in their sugar-phosphate backbone, they will be pulled toward the positively charged end of the gel.

Proteins, however, are not negatively charged; thus, want to separate proteins using gel electrophoresis, must first mix the proteins with a detergent called sodium dodecyl sulfate ( SDS ).

This treatment makes the proteins unfold into a linear shape and coats them with a negative charge, which allows them to migrate toward the positive end of the gel and be separated.

Visualizing the DNA fragments

Once the fragments have been separated, we can examine the gel and see what sizes of bands are found on it.

When a gel is stained with a DNA-binding dye and placed under UV light, the DNA fragments will glow, allowing us to see the DNA present at different locations along the length of the gel. TYPES OF ELECTROPHORESIS

1) Zone Electrophoresis : • Paper Electrophoresis • Gel Electrophoresis • Thin Layer Electrophoresis • Cellulose acetate Electrophoresis

2) Moving Boundary Electrophoresis : • Capillary Electrophoresis • Isotachophoresis • Isoelectric Focussing • Immuno Electrophoresis 03-Different types of gel electrophoresis

1. Agar or Agarose gel electrophoresis : - pulsed field gel electrophoresis For separating larger nucleic acids. 2. Polyacrylamide gel electrophoresis : - native For separating smaller nucleic acids. - SDS-PAGE for denaturing the proteins .

3. starch gel electrophoresis : Non-denatured proteins can be separated according to charge and size .

1- Agarose gel electrophoresis

Agar is a mixture of poly saccharides extracted from sea weeds. Agarose is a highly purified uncharged polysaccharide derived from agar. Agarose is chemically basic disaccharide repeating units of 3,6-anhydro-L- galactose

Agarose gel is a three-dimensional matrix formed of helical agarose molecules in supercoiled bundles that are aggregated into three- dimensional structures with channels and pores through which biomolecules can pass. The pore size may be predetermined by adjusting the concentration of agarose in the gel the higher the concentration of agarose, the smaller the pore size

Gel Structure of Agarose: Traditional agarose gels are most effective at the separation of DNA fragments between 100 bp and 25 kb. To separate DNA fragments larger than 25 kb, one will need to use pulse field gel electrophoresis The 3-D structure is held together with hydrogen bonds and can therefore be disrupted by heating back to a liquid state.

The melting temperature is different from the gelling temperature, depending on the sources, agarose gel has a gelling temperature of 35–42 °C and a melting temperature of 85– 95 °C

In this method, DNA is forced to migrate through a highly cross-linked agarose matrix in response to an electric current.

In solution, the phosphates of the DNA are negatively charged, and the molecule will therefore migrate to the positive electrode. (anode). There are factors that affect migration rate through a gel: 1. size of the DNA 2. agarose concentration 3. voltage applied 4. presence of ethidium bromide 5. conformation of the DNA 6. ionic strength of the running buffer.

This matrix creates resistance and means that smaller molecules migrate more quickly while larger molecules migrate more slowly.

The difference in migration rate is how we separate the different sizes of DNA molecule to determine their length. 03-Preparation of the Gel

Weigh out the appropriate mass of agarose into an 01 Erlenmeyer flask.

Agarose gels are prepared using a w/v percentage solution. The concentration of agarose in a gel will depend on the sizes of the DNA fragments to be separated, with most gels ranging between 0.5%- 2%.

The volume of the buffer should not be greater than 1/3 of the capacity of the flask.

Add running buffer to the agarose-containing flask. Swirl to mix. The most common gel running buffers are TAE (40 mM 02 Tris-acetate, 1 mM EDTA) and TBE (45 mM Tris-borate, 1 mM EDTA).

Melt the agarose/buffer mixture. This is most commonly done by heating in a microwave, but can also be done over a Bunsen 03 flame.

At 30 s intervals, remove the flask and swirl the contents to mix well. Repeat until the agarose has completely dissolved.

Add ethidium bromide (EtBr) to a concentration of 0.5 04 μg/ml. Alternatively, the gel may also be stained after electrophoresis in running buffer containing 0.5 μg/ml EtBr for 15-30 min, followed by destaining in running buffer for an equal length of time. EtBr is a suspected carcinogen and must be properly disposed of per institution regulations.

Gloves should always be worn when handling gels containing EtBr. Alternative dyes for the staining of DNA are available however EtBr remains the most popular one due to its sensitivity and cost.

Allow the agarose to cool either on the 05 benchtop or by incubation in a 65 °C water bath. Failure to do so will warp the gel tray.

Place the gel tray into the casting apparatus. Alternatively, one may also tape 06 the open edges of a gel tray to create a mold.

Place an appropriate comb into the gel mold to create the wells. Allow the agarose to set at room temperature. Remove the comb and place the gel in the gel box. Alternatively, the gel can also be 07 wrapped in plastic wrap and stored at 4 °C until use Program the power supply to desired voltage (1-5V/cm between 08 electrodes). Add enough running buffer to cover the surface of the gel. It is important to use the same running buffer as the one used to prepare the gel.

Buffers Buffers in gel electrophoresis are used to provide ions that carry a current and to maintain pH at a relatively constant value.

Buffers Function Barbitone Buffer( 8.0 pH) Serum protein separation • Poor resolution , weak buffer Phosphate Buffer (pH- 7.5) Enzyme separation • Low buffering capacity :-High conductivity.

Tris – borate – EDTA buffer (TBE) Nucleic acid separation • Good resolution , pH- 8.0 high buffering capacity ,low conductivity.

Tris – acetate –EDTA buffer (TAE) •Nucleic acid separation • Good resolution , pH – 8.0 high buffering capacity ,low conductivity

Tris – glycine buffer (pH8.0) Protein separation • High buffering capacity, low conductivity Add loading dye to the DNA samples to be separated , Gel 09 loading dye is typically made at 6X concentration (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol).

Loading dye helps to track how far your DNA sample has traveled, and also allows the sample to sink into the gel.

Slowly and carefully load the DNA sample(s) into the gel . 10 An appropriate DNA size marker (ladder) should always be loaded along with experimental samples.

Replace the lid to the gel box. The cathode (black leads) should be closer the wells than the anode (red leads). Double check that the 11 electrodes are plugged into the correct slots in the power supply.

Turn on the power. Run the gel until the dye has migrated to an appropriate distance.

. Observing Separated DNA fragments

Remove gel from the gel box. Drain off excess When electrophoresis buffer from the surface Remove the gel from the gel tray and has completed, turn of the gel. Place the gel expose the gel to UV light. tray on paper towels to off the power supply absorb any extra and remove the lid of running buffer This is most commonly done using a gel the gel box. documentation system , DNA bands should show up as orange fluorescent bands. Take a picture of the gel . gel documentation system.

DNA bands should show up as image of a gel post electrophoresis. orange fluorescent bands The exact sizes of separated DNA fragments can be determined by plotting the log of the molecular weight for the different bands of a DNA standard against the distance traveled by each band. The DNA standard contains a mixture of DNA fragments of pre-determined sizes that can be compared against the unknown DNA samples .

is important to note that different forms of DNA move through the gel at different rates.

Supercoiled plasmid DNA, because of its compact conformation, moves through the gel fastest, followed by a linear DNA fragment of the same size, with the open circular form traveling the slowest. I. pulsed field gel electrophoresis

Pulse Field Gel Electrophoresis (PFGE) is a powerful genotyping technique used for the separation of large DNA molecules (entire genomic DNA) after digesting it with unique restriction enzymes and applying to a gel matrix under the electric field that periodically changes direction.

PFGE is a variation of agarose gel electrophoresis that permits analysis of bacterial DNA fragments over an order of magnitude larger than that with conventional restriction enzyme analysis.

Whereas standard DNA gel electrophoresis commonly resolves fragments up to ∼50 kb in size, PFGE fractionates DNA molecules up to 10 Mb. An example of a single PFGE cycle, the arrows indicate which electrodes are active during a certain pulse cycle.

The DNA is pulled at different angles throughout the program, with the net result being the DNA moving slowly towards the bottom of the gel. comparison of PFGE versus conventional electrophoresis

In the case of PFGE, the direction of current cycles between 1, 2, and 3. unlike conventional electrophoresis where current only runs in a single direction, PFGE cycles between several directions, allowing for separation of large molecular weight DNA . Advantages of PFGE

PFGE subtyping has been successfully applied to the subtyping of many pathogenic bacteria and has high concordance with epidemiological relatedness.

PFGE has been repeatedly shown to be more discriminating than methods such as ribotyping or multilocus sequence typing for many bacteria. PFGE in the same basic format can be applied as a universal generic method for subtyping of bacteria. (Only the choice of the restriction enzyme and conditions for electrophoresis need to be optimized for each species.) DNA restriction patterns generated by PFGE are stable and reproducible. Limitations of PFGE

• Time consuming. • Requires a trained and skilled technician. • Does not discriminate between all unrelated isolates. • Pattern results vary slightly between technicians. • Can’t optimize separation in every part of the gel at the same time. Don’t really know if bands of same size are same pieces of DNA. • Bands are not independent. • Change in one restriction site can mean more than one band change. “Relatedness” should be used as a guide, not true phylogenetic measure. • Some strains cannot be typed by PFGE. APPLICATION

•PFGE may be used for genotyping or genetic fingerprinting.

•It is commonly considered a gold standard in epidemiological studies of pathogenic organisms.

•Subtyping has made it easier to discriminate among strains of Listeria monocytogenes and thus to link environmental or food isolates with clinical infections. 2- POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE)

It is prepared by polymerizing acryl amide monomers in the presence of methylene-bisacrylamide to cross link the monomers. •Structure of acrylamide (CH2=CH-CO-NH2 ) •Polyacrylamide gel structure held together by covalent cross-links. •Polyacrylamide gels are tougher than agarose gels. •It is thermostable, transparent, strong and relatively chemically inert. •Gels are uncharged and are prepared in a variety of pore sizes. •Proteins are separated on the basis of charge to mass ratio and molecular size, a phenomenon called Molecular sieving.

Vs. Agarose Polyacrylamide Gel

• Polysaccharide extracted from • Cross-linked polymer of sea weed. acrylamide. • Gel casted horizontally • Gel casted vertically. • Non-toxic. • Potent neuro-toxic • Separate large molecules • Separate small molecules. • Commonly used for DNA • Used for DNA or protein separations. separations. • Staining can be done before or • Staining can be done after pouring pouring the gel. the gel. Types of PAGE

PAGE can be classified according the separation conditions into:

I. Native-page

II. Denatured-page or sds-page

I. NATIVE-PAGE

• Native gels are run in non-denaturing conditions, so that the analyte's natural structure is maintained. • Separation is based upon charge, size, and shape of macromolecules. • Useful for separation or purification of mixture of proteins. • This was the original mode of electrophoresis. II. DENATURED-PAGE OR SDS-PAGE

• SDS polyacrylamide gel largely eliminates the influence of the structure and charge, and proteins are separated solely based the molecular weight of proteins. • The common method for determining MW of proteins. • Very useful for checking purity of protein samples. • Denaturing of protein sample is needed because protein structure is stable with hydrophobic interaction, hydrogen bond, and disulphide bond. • Stable structure does not favour electrophoresis and need to disturb its structure • Denaturing agents SDS, urea, and beta mercaptoethanol

Principle

Sodium dodecyl sulfate (SDS) is a detergent that breaks up the interactions between proteins. Urea breaks the hydrogen bonds between the base pairs of the nucleic acid, causing the constituent strands to separate. SDS is an anionic detergent that disrupts secondary and non- disulfide-linked tertiary structures and additionally applies a negative charge to each protein in proportion to its mass.

In addition to SDS, proteins may optionally be briefly heated to near boiling in the presence of a reducing agent, such as dithiothreitol (DTT) or 2-mercaptoethanol which further denatures the proteins by reducing disulfide linkages, thus overcoming some forms of tertiary protein folding, and breaking up quaternary protein structure

Differences

Native PAGE SDS PAGE

• Separation is based upon • Separation is based upon charge, size, and shape of the molecular weight of macromolecules. proteins. • Useful for separation • The most common method and/or purification of for determining MW of mixture of proteins proteins • This was the original mode • Very useful for checking of electrophoresis. purity of protein samples .

Applications of PAGE

• Used for estimation of molecular weight of proteins and nucleic acids. • Peptide mapping. • Determination of subunit structure of proteins. • Purification of isolated proteins. • Comparison of the polypeptide composition of different samples • Monitoring changes of protein content in body fluids • Identifying disulfide bonds between protein Quantifying proteins • Blotting applications Advantages of PAGE

● Stable ● Can accommodate chemically larger quantities of cross-linked ● Greater DNA without significant gel resolving loss in resolution • The pore size of the power (Sharp polyacrylamide gels

bands) can be altered in an easy and controllable fashion by changing the concentrations of the two monomers.

● Good for separation of low molecular weight fragments

Disadvantages of PAGE

• Generally more difficult to prepare and handle, involving a longer time for preparation than agarose gels. • Toxic monomers • Gels are tedious to prepare and often leak • Need new gel for each experiment Stable chemically cross-linked gel 3- STARCH GEL ELECTROPHORESIS

• ntroduced by Smithies (1955). Starch gel is one of a wide variety of supporting media that can be used for horizontal zone electrophoresis.

Such gels are prepared by heating and cooling a quantity of partially hydrolyzed starch in an appropriate buffer solution. The choice of buffer is somewhat empirical and a wide variety of compositions have been used successfully.

• 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. General procedure

01 • Starch is hydrolyzed in acetone at 37 Cͦ .

02 • Suspension neutralized with sodium acetate • Wash with distilled water and acetone 03

04 • Hydrolyzed starch is heated

05 • Cool in buffer set as gel

06 • Sample is applied to gel by soaking filter paper

07 • Insert it in the gel. • Appearance of colored band indicates the presence of active 08 enzymes . ADVANTAGES:

• High resolving power and sharp zones are obtained. • The components resolved can be recovered in reasonable yield especially proteins. • Can be used for analytical as well as preparative electrophoresis.

DISADVANTAGES:

• Electro osmotic effect. • Variation in pore size from batch to batch THANK YOU