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Why is gel used in electrophoresis?

Why is gel used in electrophoresis?


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In analysing amino acids content in a protein through gel electrophoresis, What's the purpose of the gel? Wouldn't putting the amino acid in the gel prohibit the amino acid from dissolving into the buffer solution (which its pH controls the protonation and de-protonation of the amino acid)?


In gel electrophoresis, the gel is the mechanism by which macromolecules of different sizes are separated. By loading the gel with amino acids (or proteins or DNA), you start all of the samples equally, and then push them through a gel using a salty buffer solution that is electrified.

The gel has a certain concentration of a polymer (commonly agarose or polyacrylamide) that acts as a mesh that slows your macromolecules down. So, after running a gel for a certain amount of time, molecules of different sizes will have traveled different distances. This can be visualized by different methods (ethidium bromide for DNA and antibodies for proteins, commonly) after further preparation.

So, the amino acids in question should not be dissolved in buffer, as this would result in loss of sample.

Edit:

Regarding the last point: when loading samples on a gel, they must first be dissolved in some loading buffer that will help the sample settle into the well and not diffuse out. I took your mention of "buffer" in the original question to be the running buffer for electrophoresis, which you should not dissolve your sample in. I am also unfamiliar with analyzing amino acids by electrophoresis, and I do not know if there is a specialized loading buffer that kind of analysis would require. A quick look didn't uncover anything, but you might be in a better position to answer the loading buffer issue.


Why is buffer used in gel electrophoresis instead of water?

Buffers in gel electrophoresis are used to provide ions that carry a current and to maintain the pH at a relatively constant value. These buffers have plenty of ions in them, which is necessary for the passage of electricity through them.

Additionally, what are all the purposes of the buffer? A buffer is a solution that can resist pH change upon the addition of an acidic or basic components. It is able to neutralize small amounts of added acid or base, thus maintaining the pH of the solution relatively stable. This is important for processes and/or reactions which require specific and stable pH ranges.

Also, what would happen if you use water instead of TAE buffer?

Use water instead of buffer for the gel or running buffer Agarose gels are cast and run using TAE or TBE buffer. If you do use water, your gel will melt shortly after applying voltage to the electrophoresis unit.

What is the difference between TAE and TBE buffer?

TAE (Tris-acetate-EDTA) buffer is used as both a running buffer and in agarose gel. DNA sample from TAE Buffer is suitable for this purpose, while DNA from TBE buffer is not. Borate in the TBE buffer is a strong inhibitor for many enzymes.


Gel Electrophoresis

Gel electrophoresis is the process by which we take the DNA and run an electric charge through it, therefore we can use it to compare two DNA samples, hence the name DNA fingerprinting.

Explanation:

Gel electrophoresis is basically the process by which we take the DNA, and run an electric charge through it. The DNA, being negatively charged by default, will move towards the positive side. As this happens, he DNA with lower density will travel less distance up. This can create a very unique pattern of DNA.

Now you can do this to another sample of DNA, and match it up with the frequencies of the known sample. If they are the same, then they most likely came from the same source. This is called DNA fingerprinting. This is mostly used in crime investigation, where DNA found at crime scenes is compared to DNA from suspects.

The gel doesn't, you add a dye to the DNA sample before putting it in and some dye can be seen under UV light after the gel has been treated with a chemical that activate the dye (ex: ethydium bromide)

Answer:

Gel electrophoresis is a biotechnique used to separate DNA fragments and other macromolecules, such as RNA and proteins based on their size and charge.

Explanation:

The molecules to be separated are pushed by an electrical field through a gel containing small pores. The molecules travel through these pores in the gel at a speed which is inversely related. This means that a small molecule will travel a greater distance through the gel than will a larger molecule. One end of the gel has a positive charge and the other end has a negative charge.

Structurally, DNA and RNA are negatively charged molecules, so they will be pulled toward the positively charged end of the gel. In the case of proteins, they are not purely negatively charged, so first proteins are mix with an anionic detergent called sodium dodecyl sulfate. 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.

Steps of gel electrophoresis:

When DNA, RNA and protein molecules have been separated using gel electrophoresis, bands of different sizes can be detected by UV illuminator.


3.1: Gel Electrophoresis

  • Contributed by Michael Blaber
  • Professor (Biomedical Sciences) at Florida State University

Gel electrophoresis is used to characterize one of the most basic properties - molecular mass - of both polynucleotides and polypeptides. Gel electrophoresis can also be used to determine: (1) the purity of these samples, (2) heterogeneity/extent of degradation, and (3) subunit composition.

The most common gel electrophoresis materials for DNA molecules is agarose and acrylamide.

DNA agarose gels

The electrophoretic migration rate of DNA through agarose gels is dependent upon four main parameters:

1. The molecular size of the DNA. Molecules of linear duplex DNA travel through agarose gels at a rate which is inversely proportional to the log of their molecular weight.

Example: Compare molecular mass vs. expected migration rate:

1/log (Molec. Mass)
i.e. relative Mr

Figure 3.1.1:Relative migration rate with molecular mass

2. The agarose concentration. There is an inverse linear relationship between the logarithm of the electrophoretic mobility and gel concentration.

inv log(1/Gel %) (i.e. relative Mr)

Figure 3.1.2:Relative migration rate with gel concentration

3. The conformation of the DNA.

  • closed circular DNA (form-I) - typically supercoiled
  • nicked circular (form-II)
  • linear DNA (form-III)

These different forms of the same DNA migrate at different rates through an agarose gel. Almost always the linear form (form-III) migrates at the slowest rate of the three forms and supercoiled DNA (form-I) usually migrates the fastest.

4. The applied voltage.

Range of separation of linear DNA (in kilobases)

Finally, the DNA being an acidic molecule, migrates towards the positively charged electrode (cathode).

Figure 3.1.3:Gel electrophoresis setup

DNA acrylamide Gels

Acrylamide gels are useful for separation of small DNA fragments typically oligonucleotides <100 base pairs. These gels are usually of a low acrylamide concentration (<=6%) and contain the non-ionic denaturing agent Urea (6M). The denaturing agent prevents secondary structure formation in oligonucleotides and allows a relatively accurate determination of molecular mass.

Gel Electrophoresis for Proteins

Gel electrophoresis of proteins almost exclusively utilizes polyacrylamide. The acrylamide solution usually contains two components: acrylamide and bis acrylamide. A typical value for the acrylamide:bis ratio is 19:1. The bis acrylamide is essentially a cross-linking component of the acrylamide polymer. The total acrylamide concentration in the gel affects the migration of proteins through the matrix (as with the concentration of agarose).

Protein gels are usually performed under denaturing conditions in the presence of the detergent sodium dodecyl sulfate (SDS). The proteins are denatured by heat in the presence of SDS. The SDS binds, via hydrophobic interactions, to the proteins in an amount approximately proportional to the size of the protein. Due to the charged nature of the SDS molecule the proteins thus have a somewhat constant charge to mass ratio and migrate through the gel at a rate proportional to their molecular mass, The proteins migrate towards the anode.

Range of separation of Polypeptides (in kilodaltons)

Since the SDS treatment will dissociate non-covalent protein complexes, they may thus exhibit a much lower than expected molecular mass on SDS polyacrylamide gel electrophoresis (SDS PAGE). Protein PAGE gels are usually polymerized between two glass plates and run in the vertical direction.


What is Gel Electrophoresis used for?

What is Gel Electrophoresis used for: It is a process used to get the DNA strands out from the impurities. When we get a DNA sample from an organism it also have impurities in it, so we simply ignore all those impurities and pull our DNA as pure out.

All pathology, forensic and research labs excessively use this process to get pure DNA from the samples for further research and studies. This technique always comes after Polymerase Chain Reaction which is also a very common technique used in laboratories.

As, Polymerase Chain Reaction is used to multiply DNA copies and after this reaction there are many impurities in the DNA sample that we have to ignore (like primers, taq polymerase enzyme, and nucleotides etc ) and get our DNA strands out of them, so electrophoresis is used then.

Gel Electrophoresis Materials:

Gel Electrophoresis materials are as follow:

  • Gel-Tanks
  • Agarose Gel
  • Battery
  • DNA samples (to be processed).
  • Micropippete (to transfer samples in wells)
  • Gel comb.
  • Stop-watch.

Gel Electrophoresis Process:

Gel Electrophoresis Process does not possess any rocket science there it is a simple process which includes the following steps:

  1. Make agarose gel by mixing agarose powder in water and heat it. Pour the agarose solution in the gel tank with the comb placed in it and allow to cool. When it get cooled remove the comb and You will see wells are formed in the gel.
  2. Place Your DNA sample in the wells created by comb in the gel by using micropippete.
  3. Apply battery voltage on both sides of the gel tank and the DNA will start moving towards the positive pole of the battery terminal due to the negative charge on it.
  4. After sometime notice the distance covered by DNA strands in the gel. The smaller the DNA strand the more distance it will cover while larger the DNA strand lower distance will be covered by it.
  5. Separate these DNA strands and now these are ready to process further for research or any kind of investigation.

What charge does DNA have?

What charge does DNA have is a very logical question. There’s a pure negative charge on the DNA & this is the only reason that DNA moves towards the +ve end of the battery when we apply current on both sides of the gel tank. This -ve charge is also the reason for the existence of gel electrophoresis technique.

What are the Types of Electrophoresis?

There are around 14 different types of electrophoresis which are as follow:

  • Agarose-Gel Electrophoresis
  • Paper Electrophoresis.
  • PolyAcrylamide Gel Electrophoresis (PAGE)
  • Zone Electrophoresis.
  • Temperature Gradient Gel Electrophoresis & Denaturing Gradient Gel Electrophores (TGGE) & (DGGE).
  • Zone Electrophoresis.
  • Zymogram Electrophoresis.
  • Immunoelectrophoresis.
  • Pulsed-Field Electrophoresis (PFE).
  • Capillary Electrophoresis.
  • Fluorophore Assisted Carbohydrate Electrophoresis (FACE).
  • Microchip Electrophoresis.
  • Affinity Electrophoresis.

What is Agarose?

What is Agarose: A polysaccharide obtained from seaweed. It is a linear polymer made with the repeating units of agarobiose. Agarose is a part of agar content that is an isolated component from agar by removing rest of the components. Agarose goes into gel form when mixed with water.


Gel Electrophoresis: Basics & Steps

Purpose: To separate DNA molecules according to their size. This can be done for forensic purposes, to look for disease in specific genes or paternity testing.

Brief overview: DNA is negatively charged, in order to separate it by size it is put in a solution with a current running through it that pulls the negatively charged DNA to the opposite end.

Larger pieces of DNA encounter more resistance in the solution and therefore don’t move as far as smaller segments after the same amount of time.

Once the electrical current has been run, a dye is added in order to see the bands of DNA (also known as lanes), and based on their location the length of the DNA is known (measured in base pairs).


RESULTS

Recommended Procedure

The gel box is made from a plastic storage container (14 × 20 × 5 cm, 1.6 L) and fitted with aluminum foil electrodes (1 × 25 cm four-layer folded strips) set 14 cm apart, attached with adhesive tape or glue. The electrodes are connected to five 9-V batteries in series using wires fitted with alligator clips. Gels were cast in a clean glass baking dish (9 × 13 in), where a rectangle (8.5 × 5.5 cm) was drawn with a wax crayon. Telephone brand agar (1 g) was melted using a microwave oven in 50 mL of running buffer (3 g L −1 Seachem Neutral pH Regulator). After carefully pouring the molten agar into the crayon rectangle until full, a comb made from a plastic store gift card and binder clips was used to form wells about 1 cm from the end of the gel. When the gel cooled (10–20 min), the comb was removed, the gel lifted from the casting tray, placed in the gel box with the wells close to the cathode, and submerged (to 1 cm above gel) in running buffer. DNA samples (∼10 μL) such as a standard lambda HindIII ladder (Fisher Scientific ∼$1 per lane) or genomic extract were added 1:1 in loading dye (one drop corn syrup with three drops green food coloring) using a pipetter. Electrophoresis proceeded for 1 to 1.5 hr, while observing the migration of the tracking dyes. Gels were stained submerged in a gentian violet (10,000× dilution) bath for 12 to 72 hr without shaking at room temperature. DNA bands were visualized with backlighting and photographed for further analysis.

Typical Results

The result of a typical gel separating a lambda HindIII ladder is shown in Fig. 2. Notice that discrete bands are visible after staining, each representing a differently sized DNA fragment of the ladder. Students in our classroom test were successful in visualizing DNA bands in their gels. Genomic DNA tends to form diffuse, high-molecular weight bands but also can produce more than one band. These may be of different sizes or they may be different conformations (e.g. supercoiled) of the genomic DNA that lead to changes in electrophoretic mobility. Electrophoresing for longer periods will move the DNA bands further into the gel and provide better separation. A wider range of band sizes can be obtained by first shearing genomic DNA by repetitive rapid passage through a syringe needle or by using commercially available restriction enzymes.

Scheduling and Assessment

Depending on bell schedules, it might be best to separate steps in the protocol into different blocks. In our trials, the first block was used for an activity to practice pipetting and discussions about the procedure and its rationale. The gel boxes were constructed, gels poured, loaded, and run in the second block (using a lambda HindIII ladder). The teacher removed the gels from stain later on and stored these for a third block, where the band patterns were documented and analyzed. Students were given a worksheet to complete while waiting for procedures to complete that included questions about the basis for electrophoresis, the negatively charged backbone DNA that allows for electrophoretic separation, and the determination of molecular lengths by analysis of the banding patterns. Open-ended questions, student-centered responses, appropriate wait-time and nonverbal behaviors were used to engage dialogue and draw out ideas [ 15-18 ]. Students generated a standard laboratory report and were graded according to their continual interactions throughout the lessons. Teachers also can assess students' understanding through extension projects (see below).

Student Surveys

The test class at NEM was mainly sophomores in five groups of 4–5 students each. Surveys using a Likert-type scale were completed by all 21 students, with appropriate parental and school permission (Table I). Overall, scores were 4.0 or above, indicating that students appreciated the activity and found it valuable. The scores were significantly (p < 0.05) higher than expected for every question (except Q13) using chi-square analysis and the expectation of an even distribution across the responses. Students saw the activity as connected to their coursework and up-to-date. The students felt that they actively participated, learned something valuable, and wanted to do more science investigations. The highest scores were in agreement that the activity was fun and well organized. The students were not disappointed that the activity used low-cost materials rather than laboratory equipment. Although the average responses were positive, two of the students had lower scores overall, falling just below neutral (3.0). Taken together, these survey results were significantly higher (p < 0.01) than expected and suggest that students viewed the electrophoresis activity as successful and a valuable addition to an introductory high school biology course. Content surveys previously conducted after similar electrophoretic activities using laboratory equipment at NEM and other WPS high schools showed a significant increase in content knowledge specific to the activity [ 11 ]. It must be noted that a single classroom activity is not expected, by itself, to appreciably increase performance on broad-based science assessments or overall course grades.

Question Score ± SDa a Likert-type scale: 1 = strongly disagree 2 = disagree 3 = neither agree nor disagree 4 = agree 5 = strongly agree.
1. The gel laboratory sessions were well organized 4.3 ± 0.8
2. The experimental steps were explained well 4.2 ± 0.8
3. The gel laboratory stimulated active participation 4.1 ± 0.9
4. I was motivated to see the results 4.1 ± 0.9
5. The experiment worked well for my group 4.1 ± 0.9
6. I was unhappy that we used low-cost materials 2.1 ± 1.0
7. The gel laboratory made me think creatively 3.7 ± 0.8
8. I felt like I was doing real research 3.5 ± 1.0
9. Using new laboratory equipment raises my interest in science 4.0 ± 0.9
10. The gel laboratory included up-to-date information 4.0 ± 0.9
11. My teacher could answer my questions 4.1 ± 1.1
12. The gel laboratory helped me understand important concepts 3.9 ± 0.9
13. My understanding of DNA is better after the gel laboratory 3.7 ± 1.1
14. I learned how to use micropipetters 4.0 ± 1.2
15. I feel like I learned something from this activity 4.1 ± 0.8
16. The gel laboratory was relevant to my class 4.2 ± 0.8
17. The gel laboratory was a valuable learning tool 3.9 ± 1.0
18. The activity made me want to do more experiments 3.9 ± 1.0
19. The gel laboratory was of high quality 4.0 ± 0.8
20. It was fun to do the gel laboratory 4.3 ± 0.8
  • a Likert-type scale: 1 = strongly disagree 2 = disagree 3 = neither agree nor disagree 4 = agree 5 = strongly agree.

Basic principles of gel electrophoresis to separate nucleic acids

Gel electrophoresis is a common laboratory technique in molecular biology to identify, quantify, and purify nucleic acids. Because of its speed, simplicity, and versatility, the method is widely employed for separation and analysis of nucleic acids. Using gel electrophoresis, nucleic acids in the range of approximately 0.1–25 kbp can be separated for analysis in a matter of minutes to hours, and separated nucleic acids can be recovered from the gels with relatively high purity and efficiency [1,2].

The technique involves the application of an electrical field to mixtures of charged molecules to cause them to migrate, on the basis of size, charge, and structure, through a gel matrix. The phosphate groups of the ribose-phosphate backbones of nucleic acids are negatively charged at neutral to basic pH (Figure 1A). As such, each nucleotide carries a net negative charge, meaning the overall charge of a nucleic acid molecule is proportional to the total number of nucleotides or its mass. In other words, DNA or RNA molecules carry a constant charge-to-mass ratio. As a result, their mobility in gel electrophoresis is determined mainly on the basis of size when they have comparable structure (learn more about how nucleic acid structure impacts migration). Therefore, when subjected to an electrical field, nucleic acids migrate from the negative electrode (cathode) toward the positive electrode (anode), with shorter fragments moving more rapidly than longer ones, resulting in separation based on size (Figure 1B).

Figure 1. (A) Net negative charges carried by a nucleic acid chain. (B) Separation of nucleic acid fragments of varying lengths in gel electrophoresis.

Furthermore, the migration distances of nucleic acids in gel electrophoresis generally display a predictable correlation with their sizes, enabling calculation of the size of nucleic acids in a given sample. For linear double-stranded DNA fragments, migration distance is inversely proportional to the log of the molecular weight, within a certain range (Figure 2A) [3]. For approximate sizing, migration distances are commonly compared to samples containing molecules of known sizes (molecular weight standards, sometimes referred to as “ladders”) which are often included in the gel run. A widely accepted model of nucleic acid mobility through a gel is “biased reptation”— migration biased towards the applied electrical force and involving a snaking movement where the leading edge pulls the rest (Figure 2B) [4,5]. This model has been visualized by fluorescence microscopy [6].

Figure 2. Mobility of nucleic acids in gel electrophoresis. (A) Correlation of size and migration of linear double-stranded DNA fragments. (B) Biased reptation model.


Why is gel used in electrophoresis? - Biology

BISC411
EXPERIMENTAL MOLECULAR BIOLOGY OF THE CELL

Principles of Polyacrylamide Gel Electrophoresis (PAGE)

Powerful electrophoretic techniques have been developed to separate macromolecules on the basis of molecular weight. The mobility of a molecule in an electric field is inversely proportional to molecular friction which is the result of its molecular size and shape, and directly proportional to the voltage and the charge of the molecule. Proteins could be resolved electrophoretically in a semi-solid matrix strictly on the basis of molecular weight if, at a set voltage, a way could be found to charge these molecules to the same degree and to the same sign. Under these conditions, the mobility of the molecules would be simply inversely proportional to their size.

It is precisely this idea which is exploited in PAGE to separate polypeptides according to their molecular weights. In PAGE, proteins charged negatively by the binding of the anionic detergent SDS (sodium dodecyl sulfate) separate within a matrix of polyacrylamide gel in an electric field according to their molecular weights.
Polyacrylamide is formed by the polymerization of the monomer molecule-acrylamide crosslinked by N,N'-methylene-bis-acrylamide (abbreviated BIS). Free radicals generated by ammonium persulfate (APS) and a catalyst acting as an oxygen scavenger (-N,N,N',N'-tetramethylethylene diamine [TEMED]) are required to start the polymerization since acrylamide and BIS are nonreactive by themselves or when mixed together.

The distinct advantage of acrylamide gel systems is that the initial concentrations of acrylamide and BIS control the hardness and degree of crosslinking of the gel. The hardness of a gel in turn controls the friction that macromolecules experience as they move through the gel in an electric field, and therefore affects the resolution of the components to be separated. Hard gels (12-20% acrylamide) retard the migration of large molecules more than they do small ones. In certain cases, high concentration acrylamide gels are so tight that they exclude large molecules from entering the gel but allow the migration and resolution of low molecular weight components of a complex mixture. Alternatively, in a loose gel (4-8% acrylamide), high molecular weight molecules migrate much farther down the gel and, in some instances, can move right out of the matrix.

SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Sodium dodecyl sulfate (SDS or sodium lauryl sulfate) is an anionic detergent which denatures proteins molecules without breaking peptide bonds. It binds strongly to all proteins and creates a very high and constant charge:mass ratio for all denatured proteins. After treatment with SDS, irrespective of their native charges, all proteins acquire a high negative charge.

Denaturation of proteins is performed by heating them in a buffer containing a soluble thiol reducing agent (e.g. 2-mercaptoethanol dithiothreitol) and SDS. Mercaptoethanol reduces all disulfide bonds of cysteine residues to free sulfhydryl groups, and heating in SDS disrupts all intra- and intermolecular protein interactions. This treatment yields individual polypeptide chains which carry an excess negative charge induced by the binding of the detergent, and an identical charge:mass ratio. Thereafter, the denatured proteins can be resolved electrophoretically strictly on the basis of size in a buffered polyacrylamide gel which contains SDS and thiol reducing agents.

SDS-PAGE gel systems are exceedingly useful in analyzing and resolving complex protein mixtures. Many applications and modifications of this technique are relevant to modern experimental biologists. Some are mentioned below. They are employed to monitor enzyme purification, to determine the subunit composition of oligomeric proteins, to characterize the protein components of subcellular organelles and membranes, and to assign specific proteins to specific genes by comparing protein extracts of wild-type organisms and suppressible mutants. In addition, the mobility of polypeptides in SDS-PAGE gel systems is proportional to the inverse of the log of their molecular weights. This property makes it possible to measure the molecular weight of an unknown protein with an accuracy of +/- 5%, quickly, cheaply and reproducibly.

Discontinuous SDS Polyacrylamide Gel Electrophoresis

Disc gels are constructed with two different acrylamide gels, one on top of the other. The upper or stacking gel contains 4-5% acrylamide (a very loose gel) weakly buffered at pH 9.0. The lower resolving gel (often called the running gel), contains a higher acrylamide concentration, or a gradient of acrylamide, strongly buffered at pH 9.0. Both gels can be cast as tubes in glass or plastic cylinders (tube gels), or as thin slabs within glass plates, an arrangement which improves resolution considerably, and which makes it possible to analyze and compare many protein samples at once, and on the same gel (slab gels). Today, you will be constructing and running slab gels.

The ionic strength discontinuity between the loose stacking gel and the hard running gel leads to a voltage discontinuity as current is applied. The goal of these gels is to maximize resolution of protein molecules by reducing and concentrating the sample to an ultrathin zone (1-100 nm) at the stacking gel:running gel boundary. The protein sample is applied in a well within the stacking gel as a rather long liquid column (0.2-0.5 cm) depending on the amount and the thickness of the gel or tube. The protein sample contains glycerol or sucrose so that it can be overlaid with a running buffer. This buffer is called the running buffer, and the arrangement is such that the top and bottom of the gel are in running buffer to make a circuit.

As current is applied, the proteins start to migrate downward through the stacking gel toward the positive pole, since they are negatively charged by the bound SDS. Since the stacking gel is very loose, low and average molecular weight proteins are not impeded in their migration and move much more quickly than in the running gel. In addition, the lower ionic strength of the stacking gel (weak buffer) creates a high electrical resistance, (i.e., a high electric field V/cm) to make proteins move faster than in the running gel (high ionic strength, lower resistance, hence lower electric field, V/cm). Remember that applied voltage results in current flow in the gel through the migration of ions. Hence low ionic strength means high resistance because fewer ions are present to dissipate the voltage and the electric field (V/cm) is increased causing the highly polyanionic proteins to migrate rapidly.

The rapid migration of proteins through the stacking gel causes them to accumulate and stack as a very thin zone at the stacking gel/running gel boundary, and most importantly, since the 4-5% stacking gel affects the mobility of the large components only slightly, the stack is arranged in order of mobility of the proteins in the mixture. This stacking effect results in superior resolution within the running gel, where polypeptides enter and migrate much more slowly, according to their size and shape.

In all gel systems, a tracking dye (usually Bromophenol blue) is introduced with the protein sample to determine the time at which the operation should be stopped. Bromophenol blue is a small molecule which travels essentially unimpeded just behind the ion front moving down toward the bottom of the gel. Few protein molecules travel ahead of this tracking dye. When the dye front reaches the bottom of the running gel, the current is turned off to make sure that proteins do not electrophorese out of the gel into the buffer tank.

Visualizing the Proteins

Gels are removed from tubes or from the glass plates and stained with a dye, Coomassie Brilliant Blue. Coomassie blue binds strongly to all proteins. Unbound dye is removed by extensive washing of the gel. Blue protein bands can thereafter be located and quantified since the amount of bound dye is proportional to protein content. Stained gels can be dried and preserved, photographed or scanned with a recording densitometer to measure the intensity of the color in each protein band. Alternatively, if the proteins are radioactive, the protein bands can be detected by autoradiography, a technique that is widely used in modern cell and molecular biology. When gels are prepared as thin slabs to maximize resolution as you will do today, the slabs of acrylamide are removed from the support glass plates and dried on filter paper. A piece of X-ray film is placed and clamped tight over the dried slab in a light-proof box. The X-ray film is exposed by the radioactivity in the protein bands and, after developing, dark spots or bands can be seen on the film. These dark bands can in turn be quantified since their intensity is proportional to the amount of radioactivity and hence to protein content.


Restriction Enzymes

A DNA fingerprint is created by first digesting a DNA sample with a restriction enzyme. Restriction enzymes recognize very specific DNA sequences (such as 5’-GAATTC-3’), which are usually palindromes. Palindromic sequences allow the same sequence to be recognized on both strands of DNA (Figure 5, top). The restriction enzyme will then cut the DNA at a specific point. You can think of restriction enzymes as very specific molecular scissors. In the top DNA sequence seen in Figure 5, the restriction enzyme EcoRI recognizes the sequence GAATTC and cuts between the G and the A on both strands of DNA.

A point mutation (a change in one base in the DNA) can change the site a restriction enzyme recognizes and eliminate the restriction site (Figure 5, bottom). The restriction enzyme will not cut the DNA since its recognition site is no longer present. Alternatively, the point mutation could create a new restriction site where there was not one originally. If the restriction site is present, two short fragments will be produced. In contrast, if the restriction site is absent, one long fragment will remain intact. Other than identical twins, no two individuals will have the same DNA fingerprint. The genomes of any two non-identical individuals will contain a large number of differences in the DNA sequence that have the potential to change restriction sites.

Figure 5 In the top sequence (above the blue line), the EcoR1 restriction enzyme recognition site (GAATTC) is present, so the enzyme cuts the DNA between the G and the A on both strands of DNA. This produces two shorter segments of DNA. In the bottom sequence (below the blue line), a point mutation (red) has eliminated the recognition sequence so the enzyme does not cut the DNA. Credit: Lisa Bartee CC 4.0


How gel concentration affects relative mobility

So what happens if you want to characterize all of the proteins in a sample? . run more than one gel, of course! A gel with low density will resolve the larger polypeptides while cutting off the lighter ones, and one of higher density will reveal the smaller polypeptides, while compressing and possibly distorting the larger ones.

Here are some examples of the effect of acrylamide concentration on relative mobility. Molecular weight standards are identified by number. A typical erythrocyte membrane protein sample is also presented, with band 3 protein labeled as a reference.

Standard 1 = myosin (205,000) std. 2 = beta-galactosidase (116,000) std. 3 = phosphorylase B (92,000) std. 4 = bovine serum albumin (66,000) std. 5 = egg albumin (45,000) std. 6 = carbonic anhydrase (29,000).

On the gels from 6 to 10% there is a distinct dark doublet at the top of the membrane lane. Notice in the 12% gel the doublet is jammed together and appears as one band. One could estimate MW of band 3 from the first five gels although the best estimate comes from the 6%, which produced the greatest separation between the standards on either side of band 3. The 12% gel did not resolve bands well at all above the fourth standard (serum albumin, 66,000). Analysis should be confined to the part of the gel below 66,000 or even lower if the same sample was run on a lower density gel.

Frequently, students analyze the upper part of a high density acrylamide gel that overlaps part of a lower density gel. That practice suggests that the student missed the point of the anaysis and/or did not understand the limitations of the method. Interpret only that part of the denser gel that doesn't overlap the other one. To put it another way, use high density gels to study proteins (or parts of proteins) of relatively low molecular weight, and lower density gels to resolve proteins of higher molecular weight.

Don't forget that different polypeptides can have similar or even identical molecular masses. One band on a gel can therefore consist of one or more polypeptides. This is most likely to happen toward the top of a gel, and especially in higher density gels.


Watch the video: Γνωριμία με το Gel - meet gel (May 2022).