Electrophoresis1

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1) Electrophoresis - IntroductionIntroduction

Electrophoresis is the migration or separation of charged particles or solutes of a liquid solution

in an electrical field. Conventional electrophoresis is tedious and time consuming.Electrophoresis automation and newer electrophoresis techniques have revitalized the utilizationof electrophoresis in today's clinical laboratories. Molecular diagnostic analysis using

electrophoresis and research in proteomics have also contributed to this revitalization.

Principle of Electrophoresis

Charged particles under the influence of a liquid media placed in an electric field will migrate to

the electrode of the opposite charge. Positive ions (cations) will migrate to the cathode, thenegative electrode. Negative ions (anions) will migrate to the anode, the positive electrode.

Electrophoresis can be one dimensional (i.e. one plane of separation) or two dimensional. Onedimensional electrophoresis is used for most routine protein and nucleic acid separations. Twodimensional separation of proteins is used for finger printing , and when properly constructed

can be extremely accurate in resolving all of the proteins present within a cell (greater than1,500).

The support medium for electrophoresis can be formed into a gel within a tube or it can belayered into flat sheets. The tubes are used for easy one dimensional separations (nearly anyone

can make their own apparatus from inexpensive materials found in any lab), while the sheetshave a larger surface area and are better for two- dimensional separations.

When the detergent SDS (sodium dodecyl sulfate) is used with proteins, all of the proteinsbecome negatively charged by their attachment to the SDS anions. When separated on apolyacrylamide gel, the procedure is abbreviated as SDS--PAGE (for Sodium Dodecyl Sulfate

PolyAcrylamide Gel Electrophoresis). The technique has become a standard means for molecularweight determination.

Polyacrylamide gels are formed from the polymerization of two compounds, acrylamide and N,N

-methylene- bis-acrylamide (Bis, for short). Bis is a cross-linking agent for the gels. Thepolymerization is initiated by the addition of ammonium persulfate along with either -dimethyl

amino-propionitrile (DMAP) or N,N,N ,N ,- tetramethylethylenediamine (TEMED). The gelsare neutral, hydrophillic, three-dimensional networks of long hydrocarbons crosslinked by

methylene groups.

The separation of molecules within a gel is determined by the relative size of the pores formed

within the gel. The pore size of a gel is determined by two factors, the total amount ofacrylamide present (designated as %T) and the amount of cross-linker (%C). As the total amount

of acrylamide increases, the pore size decreases. With cross- linking, 5%C gives the smallestpore size. Any increase or decrease in %C increases the pore size. Gels are designated as percent

solutions and will have two necessary parameters. The total acrylamide is given as a % (w/v) of


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the acrylamide plus the bis-acrylamide. Thus, a 7 1/2 %T would indicate that there is a total of7.5 gms of acrylamide and bis per 100 ml of gel. A gel designated as 7.5%T:5%C would have a

total of 7.5% (w/v) acrylamide + bis, and the bis would be 5% of the total (with pure acrylamidecomposing the remaining 2.5%).

Proteins with molecular weights ranging from 10,000 to 1,000,000 may be separated with 71/2% acrylamide gels, while proteins with higher molecular weights require lower acrylamidegel concentrations. Conversely, gels up to 30% have been used to separate small polypeptides.

The higher the gel concentration, the smaller the pore size of the gel and the better it will be ableto separate smaller molecules. The percent gel to use depends on the molecular weight of the

protein to be separated. Use 5% gels for proteins ranging from 60,000 to 200,000 daltons, 10%gels for a range of 16,000 to 70,000 daltons and 15% gels for a range of 12,000 to 45,000

daltons. 3

Cationic vs anionic systems

In electrophoresis, proteins are separated on the basis of charge, and the charge of a protein canbe either + or -- , depending upon the pH of the buffer. In normal operation, a column of gel ispartitioned into three sections, known as the Separating or Running Gel, the Stacking Gel and the

Sample Gel. The sample gel may be eliminated and the sample introduced via a dense non-convective medium such as sucrose. Electrodes are attached to the ends of the column and an

electric current passed through the partitioned gels. If the electrodes are arranged in such a waythat the upper bath is -- (cathode), while the lower bath is + (anode), and -- anions are allowed to

flow toward the anode, the system is known as an anionic system. Flow in the opposite direction,with + cations flowing to the cathode is a cationic system.

Tube vs Slab Systems

Two basic approaches have been used in the design of electrophoresis protocols. One, column

electrophoresis, uses tubular gels formed in glass tubes, while the other, slab gel electrophoresis,uses flat gels formed between two plates of glass. Tube gels have an advantage in that the

movement of molecules through the gels is less prone to lateral movement and thus there is aslightly improved resolution of the bands, particularly for proteins. It is also more economical,

since it is relatively easy to construct homemade systems from materials on hand. However, slabgels have the advantage of allowing for two dimensional analysis, and of running multiple

samples simultaneously in the same gel.

Slab gels are designed with multiple lanes set up such that samples run in parallel. The size and

number of the lanes can be varied and, since the samples run in the same medium, there is lesslikelihood of sample variation due to minor changes in the gel structure. Slab gels are

unquestionably the the technique of choice for any blot analyses and for autoradiographicanalysis. Consequently, for laboratories performing routine nucleic acid analyses, and those

employing antigenic controls, slab gels have become standard. The availability of reasonablypriced commercial slab gel units has increased the use of slab gel systems, and the use of tube

gels is becoming rare.


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The theory and operation of slab gel electrophoresis is identical to tube gel electrophoresis.Which system is used depends more on the experience of the investigator than on any other

factor, and the availability of equipment.

2) Types of electrophoresis

There are quite a number of types of electrophoresis commonly used. Some of them are listed below:

y SDS-PAGE.y Native Gels.y Electrofocusing Gels.y DNA Agarose Gels.y DNA denaturing polyacrylamide gels (often called sequencing gels).y Capillary electrophoresis.y Pulse fieldy 2-D lectrophoresis.

a) Pulsed Field Gel ElectrophoresisConventional methods of gel electrophoresis are carried out by placing DNA samples in a solid

matrix (agarose or polyacrilamide) and inducing the molecules to migrate through the gel under astatic electric field. When DNA molecules are under the influence of this electric field, they

elongate and align themselves with the field, migrating toward the anode in a process calledreptation. There are several parameters that affect the migration of DNA through the gel:

concentration and composition of the gel, the buffer, the temperature, and the voltage gradient ofthe electric field. In DNA electrophoresis by the standard method however, DNA molecules

larger than 20kb show essentially the same mobility in a static electric field, making

differentiation between these DNA molecules impossible. The first attempts to resolve theselarger fragments included using low percentage agarose gels and low voltage gradients. Evenunder these extreme conditions, separation of large DNA molecules was difficult. In 1984, David

Schwartz was able to offer a new technique. He suggested that periodically changing theorientation of the electric field would force DNA molecules in the gel to relax upon the removal

of the first field and elongate to allign with the new field. It was his assumption that this processshould be size dependent. Schwartz was finally able to demonstrate the effectiveness of this

technique when he successfully separted yeast chromosomes that were several hundred kilobasesin length. (Birren et al., 1993)

Development of the Technique

The method of pulsed field gel electrophoresis was first utilized in 1982, and since then severalapparatuses have been developed for separating large molecules of DNA, all using multiple

electric fields. All systems seaparte DNA molecules within the same size range but differ in thespeed of separation and the resolution.

The first PFGE apparatus used two alternating electric fields, one homogeneous and the other

nonhomogeneous. The OFAGE apparatus uses two nonhomogeneous electric fields and was


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developed shortly after PFGE was developed. A problem with these first two methods arose due

to the fact that the DNA molecules ran in a curved trajectory, making lane-to-lane comparisons

difficult. Thus, the TAFE system used homogeneous electric fields produced across the width of

the gel to eliminate the "bent" lanes. This system also has its drawbacks because the reorientation

angles are not constant throughout the gel, thus molecules do not move at constant velocity

throughout the gel and liquid samples cannot be used. A modification of the TAFE system is

found in the ST/RIDE system which allows for the changing of reorientation angles while the gel

is running. This technique minimizes band stacking and liquid samples can be used. The FIGE

apparatus is the simplest to construct and operates by periodically inverting a uniform electric

field in one dimension. Another more complex approach using the same method is ZIFE or zero

integrated field electrophoresis. ZIFE is slower than FIGE but has the ability to resolve larger

molecules of DNA. The advantage of the CHEF apparatus is that it is capable of separating a

large number of DNA samples in straight lines by generating homogeneous electric fields using

multiple electrodes arranged around a closed contour. Finally, in the crossed field apparatus, the

gel is simply placed on a moblile platform that can be rotated in order to change the orientationof the electric field relative to the DNA. (Birren et al., 1993)

Applications of PFGE

y Pulsed field gel electrophoresis has been used as a means of identifying the geneticdefects that cause many hereditary diseases. In principle, detection of chromosomalrearrangements should be easy since, when run on a gel, they produce size differences

when the normal gene and the defective gene are compared. However, when using thestandard method of the gel electrophoresis, only size differences up to 30kbp can be

detected. Below is a figure that demonstrates the difference between the hybridization

pattern of a conventional gel analysis and the hybridization pattern of a PFGE gelanalysis (Mathew, 1991).

y From December 1994 to January 1995 Salmonella agona infections increaseddramatically in England and Wales. It was necessary to characterize those who fell victimto the outbreak through genotipic methods and PFGE was the best method to provide a

genotypic fingerprint of each patient.

b) two-dimensional (2-D) electrophoresisTwo-dimensional electrophoresis (2-D electrophoresis) is a powerful and widely used method for the

analysis of complex protein mixtures extracted from cells, tissues, or other biological samples. This

technique sorts proteins according to two independent properties in two discrete steps: the first-dimensionstep, isoelectric focusing (IEF), separates proteins according to their isoelectric points (pI); the second-

dimension step, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), separates proteins according to

their molecular weights (Mr, relative molecular weight). Each spot on the resulting two-dimensional array

corresponds to a single protein species in the sample. Thousands of different proteins can thus be

separated, and information such as the protein pI, the apparent molecular weight, and the amount of each

protein is obtained.


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Two-dimensional electrophoresis was first introduced by P. H. O'Farrell (1) and J. Klose (2) in 1975. In

the original technique, the first-dimension separation was performed in carrier ampholyte-containing

polyacrylamide gels cast in narrow tubes. The power of 2-D electrophoresis as a biochemical separation

technique has been recognized virtually since its introduction. Its application, however, has become

significant only in the last few years as a result of a number of developments. A large and growing

application of 2-D electrophoresis is "proteome analysis." Proteome analysis is "the analysis of the entirePROTEin complement expressed by a genOME" (6,7). The analysis involves the systematic separation,

identification, and quantification of many proteins simultaneously from a single sample. Two-

dimensional electrophoresis is used in this technique due to its unparalleled ability to separate thousands

of proteins simultaneously. Two-dimensional electrophoresis is also unique in its ability to detect post-

and co-translational modifications, which cannot be predicted from the genome sequence. Applications of

2-D electrophoresis include proteome analysis, cell differentiation, detection of disease markers,

monitoring therapies, drug discovery, cancer research, purity checks, and microscale protein purification.

3) Extraction of proteins and preparation of sample.It is actually very easy to isolate proteins and requires very few steps compared to isolationof DNA or RNA. You do need to be careful when isolating proteins as the tissue and proteins

need to be kept cold to prevent them from degrading. We will also have some proteases

inhibitors in our proteins to prevent their degradation over the next few weeks as even in the

freezer proteins can degrade over time.

PROCEDURE

0. To 10 ml of QB buffer which contains 100mM potassium phosphate buffer pH= 7.8, 1mM

EDTA, 1% Triton-X-100, and 10% glycerol add 1 vial of protease inhibitors.

1. To isolate your protein, place 1g of fresh plant tissue that has been finely cut with a razor

blade into a mortar with approximately 2 ml of cold extraction buffer and a pinch of clean sand.Grind until smooth with a pestle. It is very important to grind the tissue well. You may need to

add more buffer but the final product should be about the concistency of slightly waterytoothpaste. Keep the mortar and pestle and plant tissue as cold as possible during this process.

2. Transfer 1 ml of slurry into a 1.5 ml microfuge tube using a rubber policeman. Place the tube

on ice until you have all of your samples ready.

3. Rinse mortar and pestle (and any other paraphernalia that came into contact with the sample)to

remove all traces of sample, make sure plant tissue to be used for DNA isolation next week iskept cold (place in bag in -80 freezer.) and proceed to the protein isolation of the next plantsample.

4. Spin your samples at top speed in a microfuge at 4 degrees C for 15 minutes.

5. Transfer the liquid supernatant into a second (new) microfuge tube.


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6. Sometimes excess tissue is transferred over into the second microfuge tube. If this is the case,spin a second time for about 10 minutes and transfer this supernatant into a third microfuge tube.

7. Store the samples in ice only until you have finished your DC protein Assay, then freeze the

samples in liquid nitrogen and put them in the -80 degree freezer until we are ready to use them

on a Western Blot.

8.Set up for the DC Assay. You can do this while your samples are being spun down. This assay

will show a color change dependent on the amount of protein present in the sample. The assay istime and temperature dependent and thus the samples need to be run right along side of the

samples and you do need to keep some thought to how you time the entering of each ingrediantinto the assay for best results.

Sample Preparation for SDS-PAGE Electrophoresis

Standard

2X

Sample

Buffer:

0.5M Tris-HCl, pH 6.8

4.4% SDS

300mM Mercaptoethanol

10mg/ml Bromphenol

Blue

Mix sample with an equal volume of 2X sample buffer (For greater reproducibility, employNational Diagnostics preformulated 2X sample buffer, Protein Loading Buffer Blue 2X). Bring

to 95 C for 10 minutes, cool to room temperature before loading. If particulate is present,centrifuge samples 5 minutes at 14k RPM in microcentrifuge, and load the gel.

This protocol is sufficient for most tissue culture cells, fluid samples (such as serum andcerebrospinal fluid), bacteria and some soft tissues. For more highly structured samples, use the

following buffer:

CHES

Sample

Buffer:

1% CHES pH to

9.5 with NaOH

2% SDS

1% DTT

10% glycerol

Store @ -20 C

up to 6 months.

Homogenize samples in 5 - 15 volume of CHES buffer in a dounce homogenizer @ RT, 25 - 50strokes.


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Heat homogenate to 95C for 5 minutes and allow to cool. (homogenized samples will containsubstantial amounts of cellular debris, which must be removed by centrifugation to avoid

clogged wells. Centrifuge samples @ 14K RPM in microcentrifuge, 15 minutes.

NOTES ON YEAST AND BACTERIA: these organisms may be encapsulated in a layer of

lipopolysaccharide (LPS) which will require enzymatic digestion with Lysozyme or Zymolyaseprior to homogenization.

4) Extraction of nucleic acids or DNA and sample preparation.

Simply put, DNA Extraction is the removal of deoxyribonucleic acid (DNA) from the cells or

viruses in which it normally resides. Extraction of DNA is often an early step in many diagnostic

processes used to detect bacteria and viruses in the environment as well as diagnosing disease

and genetic disorders. These techniques include but are not limited to -

y Fluorescence In Situ Hybridization (FISH): FISH is a molecular technique that is used,among other things, to identify and enumerate specific bacterial groups.

y Terminal Restriction Fragment Length Polymorphism (T-RFLP): T-RFLP is used toidentify, characterize, and quantify spatial and temporal patterns in marine

bacterioplankton communities.y Sequencing: Portions of, or whole genomes may be sequenced as well as extra

chromosomal elements for comparison with existing sequence in the public data base.

Outline of a basic DNA Extraction -

1. Break open (lyse) the cells or virus containing the DNA of interest-This is often done by sonicating or bead beating the sample. Vortexing with phenol

(sometimes heated) is often effective for breaking down protienacious cellular walls orviral capsids. The addition of a detergent such as SDS is often necessary to remove lipid

membranes.2. DNA associated proteins, as well as other cellular proteins, may be degraded with the

addition of a protease. Precipitation of the protein is aided by the addition of a salt suchas ammonium or sodium acetate. When the sample is vortexed with phenol-chloroform

and centrifuged the proteins will remain in the organic phase and can be drawn offcarefully. The DNA will be found at the interface between the two phases.

3. DNA is the precipitated by mixing with cold ethanol or isopropanol and thencentrifuging. The DNA is insoluble in the alcohol and will come out of solution, and the

alcohol serves as a wash to remove the salt previously added.4. Wash the resultant DNA pellet with cold alcohol again and centrifuge for retrieval of the

pellet.5. After pouring the alcohol off the pellet and drying, the DNA can be re-suspended in a

buffer such as Tris or TE.


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6. Presence of DNA can be confirmed by electrophoresing on an agarose gel containingethidium bromide, or another fluorescent dye that reacts with the DNA, and checking

under UV light.

Instrumentation used in DNA Extraction -

This bead beateris used in the breaking apart or "lysing" of cells in the early steps of extractionin order to make the DNA accessible. Glass beads are added to an eppendorph tube containing a

sample of interest and the bead beater vigorously vibrates the solution causing the glass beads tophysically break apart the cells. Other methods used for lysing cells include a french press and a

sonication device.

A centrifuge such as this can spin at up to 15,000 rpm to facilitate separation of the different

phases of the extraction. It is also used to precipitate the DNA after the salts are washed awaywith ethanol and or isopropanol.

Agel boxis used to separate DNA in an agarose gel with an electrical charge. When the red and

black leads are plugged into a power supply the DNA migrates through the gel toward the

positive charge due to the net negative charge of the molecule. Different sized pieces of DNA

move at different rates, with the larger pieces moving more slowly through the porus medium,

thereby creating a size separation that can be differentiated in a gel.

5) Assembly, loading and running of gelsCassettes should be rinsed free of any excess liquid, leaving the combs in place. If casting standsare used, the clay is scraped off of the front cover and the cover removed. Gel cassettes are

separated with the aid of a single edged razor blade if necessary (having beveled plates helps).After scraping off any excess stacking gel, the surfaces of the plates must be rinsed and dried,and the best gels selected. Small air spaces may appear between stacking gel and resolving gel or

between the gel and the glass plates. As the outside pressure on the plates is relieved the glassexpands, creating some spaces. As long as there is no continuous channel from the top to the

bottom of the gel, the spaces will not influence protein migration.

The assembly of a gel running stand varies with the type of apparatus. The top of the cassettemust be continuous with an upper buffer chamber and the bottom must be continuous with a

lower chamber so that current will run through the gel itself. The cassette must be sealed in placeusing gaskets or a sealant such as agarose. In a teaching lab the assembly is best described by

going through the procedure, using a film, and/or having a demonstration set up. We fill both theupper and lower buffer compartments with an electrode buffer (running buffer) consisting of 25

mM Tris, 192 mM glycine, 0.1% sodium dodecyl sulfate (electrode buffer composition is part ofthe Laemmli method). We do not adjust pH of the electrode buffer. We remove the comb from

the gel before filling the upper buffer compartment.


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Loading gels

Hamilton syringes work well for loading samples into the wells. Ideally, the glycerol in a samplecauses it to sink neatly to the bottom of the well, allowing as much as 20 l or even more to be

loaded. If the combs do not fit well or the plates are not clean the sample often hangs up, and we

are limited to 10 l or so.

Running gels

The anode (+ electrode) must be connected to the bottom chamber and the cathode to the top

chamber. The negatively-charged proteins will move toward the anode, of course. Gels areusually run at a voltage that will run the tracking dye to the bottom as quickly as possible without

overheating the gels. Overheating can distort the acrylamide or even crack the plates. Thevoltage to be used is determined empirically. We run our gels at 150 volts.

Notes on gel assembly and running

y Criteria for a good gel include straight spacers, top, bottom of separating gel parallel, straightwells, appropriate depth of stacking gel.

y Agarose will not stick to wet surfaces, so plates and apparatus must be completely dry beforesealing; bubbles in agarose will eventually cause leaks.

y Agarose alone will not hold a gel in place - the cassette must be secured in place.y We have found that the lane dividers are less likely to be distorted if we remove combs before the

upper chamber is filled.y Lanes become distorted if the samples sit in the wells for too long before running.y The gel can't be rescued if the voltage is run backwards for any significant length of time.y If the upper chamber leaks out, the gel can be 'rescued' provided samples have entered the gel -

the cassette is removed, everything dried, cassette re-sealed in place, buffer re-added, and

electrophoresis resumed.y The apparatus should be placed in a tray in case of leaks, and not touched while the voltage is on.

6) Staining/ visualizationDisassembly and staining

When the dye front is nearly at the bottom of the gel it is time to stop the run. For low percentgels with a tight dye front, the dye should be on the verge of running off the gel. When the

percent acrylamide is high the dye front may be diffuse, since the dye is not homogeneous. If

you know the approximate position of the lowest protein band you can let the dye run off. Onlyexperience will tell you when it is appropriate to stop the run. Before removing gels the powermust be turned off and cables removed (using one hand, to avoid making a circuit).

Removal the gel from the cassette is better demonstrated than described. The plates are separated

and the gel is dropped into a staining dish containing deionized water. After a quick rinse, thewater is poured off and stain added. Staining usually requires incubation overnight, with

agitation.


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Staining protein gels

a) ProteinsA commonly used stain for detecting proteins in polyacrylamide gels is 0.1% Coomassie Blue dye in 50%

methanol, 10% glacial acetic acid. Acidified methanol precipitates the proteins. Staining is usually done

overnight with agitation. The agitation circulates the dye, facilitating penetration, and helps ensure

uniformity of staining.

The dye actually penetrates the entire gel, however it only sticks permanently to the proteins.Excess dye is washed out by 'destaining' with acetic acid/methanol, also with agitation. It is mostefficient to destain in two steps, starting with 50% methanol, 10% acetic acid for 1-2 hours, then

using 7% methanol, 10% acetic methanol to finish. The first solution shrinks the gel, squeezingout much of the liquid component, and the gel swells and clears in the second solution. Properly

stained/destained gels should display a pattern of blue protein bands against a clear background.The gels can be dried down or photographed for later analysis and documentation.

The original dye front, consisting of bromphenol blue dye, disappears during the process. In fact,

bromphenol blue is a pH indicator which turns light yellow under acid conditions, prior to beingwashed out. In low percentage gels, sufficient protein may run with the dye front so that the

position of the bromphenol blue front is permanently marked with unresolved proteins, oftenforming a continuous "front" across the bottom of the gel. In higher % gels, a distinct dye front is

usually not obtained.

Coomassie blue may not stain some proteins, especially those with high carbohydrate content.

Stains such as periodic acid-Schiff (PAS), fast green, or Kodak 'Stain's all' may detect differentpatterns. Silver staining is generally used when detection of very faint proteins is necessary.

Routine staining with Coomasie Blue is straightforward - about the only ways to ruin a gel at this

point are physical damage (ripping the gel, for example), letting dye pool and precipitate in thegel, forgetting the alcohol at some step, allowing protein to dissolve and diffuse out of the gel. If

that happens, the information is lost.

b) DNAWhen adequate migration has occured, DNA fragments are visualized by staining with ethidium

bromide. This fluorescent dye intercalates between bases of DNA and RNA. It is often incorporated into

the gel so that staining occurs during electrophoresis, but the gel can also be stained after electrophoresis

by soaking in a dilute solution of ethidium bromide. To visualize DNA or RNA, the gel is placed on a

ultraviolet transilluminator. Be aware that DNA will diffuse within the gel over time, and examination or

photography should take place shortly after cessation of electrophoresis.

c) Peroxidase


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A procedure was developed for a rapid double staining of peroxidase and other proteins in thesame polyacrylamide gels using guaiacol and Coomassie blue. The distinguishable colored bands

of peroxidase isozymes and proteins are stable for at least 8 months.

d) EsterasesIsoenzyme bands were visualized by staining with naphthol ester as substrate and coupling to an azo dye.

Staining intensities of isoenzymes were quantified by densitometric scanning.

e) dihydrouracil dehydrogenaseA rapid and sensitive method for the location of dihydrouracil dehydrogenase after disc gelelectrophoresis based on the reduction of nitroblue tetrazolium to form an insoluble dye has been

developed. Semiquantitative evaluation of enzyme activity was achieved by means ofdensitometer tracings of stained gels. The method permits detection of enzyme activity in

partially purified extracts after a minimal number of purification steps. Two enzyme bands,differing mainly in charge, were separated giving the first indication that this enzyme may

possibly exist in at least 2 isoenzyme forms in liver.

7) Discussion of electrophoresis as analytical technique in biology. Compare with other techniques

Electrophoresis may be the main technique for molecular separation in today's cell biologylaboratory. Because it is such a powerful technique, and yet reasonably easy and inexpensive, it

has become commonplace. In spite of the many physical arrangments for the apparatus, andregardless of the medium through which molecules are allowed to migrate, all electrophoretic

separations depend upon the charge distribution of the molecules being separated.

References:


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1. Guidelines given by John A. Smith, in Current Protocols in Molecular Biology, SectionII, 10.2.2 "Electrophoretic Separation of Proteins". Greene Publishing Associates,

Brooklyn 1987.2. Mary Ellen Koenn, MS, MT(ASCP), CLS(NCA), Reviewer: Leslie Lovett, MS,

MT(ASCP), Electrophoresis.

a.

http://www.medialabinc.net3. Dr. William H. Heidcamp, Cell Biology Laboratory Manual Electrophoresis Introduction,Biology Department, Gustavus Adolphus College,

St. Peter, MN56082 -- [email protected]. http://homepages.gac.edu/~cellab/chpts/chpt4/intro4.html

4. Birren, Bruce and Lai, Eric.Pulsed Field Gel Electrophoresis: A Practical Guide. San Diego,California: Academic Press Inc. 1993.http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Cobain/method.html

5. Tom Berkelman and Tirra Stenstedt, 2-D Electrophoresis: Principles and Methods 80-6429-60a. Edition AC.Technical University of Munich, August 1998

6. http://csm.jmu.edu/biology/courses/bio480_580/mblab/protein_isolation.htm7. http://www.nationaldiagnostics.com/article_info.php/articles_id/528. George Rice, ([email protected]), Rice UniversityDates ,DNA Extraction. Montana

State Universitya. http://serc.carleton.edu/microbelife/research_methods/genomics/dnaext.html

9. http://www.ruf.rice.edu/~bioslabs/studies/sds-page/gellab2b.html10.Agarose gel electrophoresis of DNA

a. http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/agardna.html 11. Ronald O. Hallock2 and Esther W. Yamada, 22 May 1973,Visualization of dihydrouracil

dehydrogenase activity after disc gel electrophoresis, Department of Biochemistry,

University of Manitoba, Winnipeg, Manitoba, Canada R3E OW3

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Electrophoresis1