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What is electrophoresis?

Electrophoresis is a laboratory equipment commonly used in the laboratory to separate charged molecules such as DNA by size.

  • The charged molecules move through a gel through which an electric current passes.
  • An electric current is applied across the gel so that one end of the gel has a positive charge and the other end has a negative charge.
  • The motion of charged molecules is called migration. Molecules move in opposite directions. Thus a negatively charged molecule is pulled towards the positive end (opposites are absorbed!).
  • The gel is made of a permeable matrix that is a bit like a sieve that molecules can pass through when an electric current passes through it.
  • Smaller molecules migrate faster than gels and therefore move more than larger parts, which migrate more slowly and therefore travel shorter distances. As a result, the molecules are separated by size.
 

Gel electrophoresis technique

Electrophoresis is a very versatile technique that basically applies an electric current to biological molecules, whether they are usually DNA, they can also be proteins or RNAs, and it breaks these pieces into larger or smaller pieces.

Used in various applications. Everything from forensics to identifying people who may have been involved in a crime, by linking their DNA pattern, their electrophoresis pattern, to a sample in a database.

The whole basis on which the human genome was built is a capillary electrophoresis that separates DNA into shorter pieces and then runs them on these electrophoresis gels, allowing the As, Cs, Ts and Gs patterns to light up.

They are also very important in protein research and then genetic mutation research. Because when proteins or DNA mutate, they often become longer or shorter, and therefore appear different from normal in gel electrophoresis, many diagnostic tests are still performed using electrophoresis, so This is a very widely used research method.

It is important to understand the function of genes and proteins, but it has now also entered the field of clinical diagnosis and forensics. Electrophoresis is usually done in a box with a positive charge on one side and a negative charge on the other.

And as we all learned in basic physics, when you place a charged molecule in an environment like that, the negative molecules move toward the positive charge, and vice versa. When you look at the proteins in the gel, in one of these boxes, you usually take the whole protein and look at the whole length of the protein again and see how big it is, and the bigger it is, the shorter it migrates to the gel, so that Small proteins are placed at the end of the gel because they have migrated.

The farthest and the largest remain at the peak. In the case of DNA, DNA is a very long molecule, so in most cases you do not want to run a complete DNA molecule from one cell on one gel.

It’s so big that it never enters the gel, so what scientists are doing, and what people are doing in the classroom today, is shredding that DNA using things like inscription enzymes that can make DNA into more controllable parts in a way Repeat and then those pieces, depending on how big the pieces are, more or less migrate into the father gel from the bottom of the box from top to bottom.

It migrates shorter into the gel, so that small proteins are placed at the bottom of the gel, because they have migrated at the farthest distances and the largest remain at the top.

In the case of DNA, DNA is a very long molecule, so in most cases you do not want to run a complete DNA molecule from one cell on one gel. It’s so big that it never enters the gel, so what scientists are doing, and what people are doing in the classroom today, is shredding that DNA using things like inscription enzymes that can make DNA into more controllable parts in a way Repeat and then those pieces, depending on how big the pieces are, more or less migrate into the father gel from the bottom of the box from top to bottom.

It migrates shorter into the gel, so that small proteins are placed at the bottom of the gel, because they have migrated at the farthest distances and the largest remain at the top.

In the case of DNA, DNA is a very long molecule, so in most cases you do not want to run a complete DNA molecule from one cell on one gel.

It’s so big that it never enters the gel, so what scientists are doing, and what people are doing in the classroom today, is shredding that DNA using things like inscription enzymes that can make DNA into more controllable parts in a way Repeat and then those pieces, depending on how big the pieces are, more or less migrate into the father gel from the bottom of the box from top to bottom.

Because they have migrated to the farthest and the greatest will remain at the peak. In the case of DNA, DNA is a very long molecule, so in most cases you do not want to run a complete DNA molecule from one cell on one gel.

It’s so big that it never enters the gel, so what scientists are doing, and what people are doing in the classroom today, is shredding that DNA using things like inscription enzymes that can make DNA into more controllable parts in a way Repeat and then those pieces, depending on how big the pieces are, more or less migrate into the father gel from the bottom of the box from top to bottom.

Because they have migrated to the farthest and the greatest will remain at the peak. In the case of DNA, DNA is a very long molecule, so in most cases you do not want to run a complete DNA molecule from one cell on one gel.

It’s so big that it never enters the gel, so what scientists are doing, and what people are doing in the classroom today, is shredding that DNA using things like inscription enzymes that can make DNA into more controllable parts in a way Repeat and then those pieces, depending on how big the pieces are, more or less migrate into the father gel from the bottom of the box from top to bottom.

A complete DNA molecule from a cell to a gel. It’s so big that it never enters the gel, so what scientists are doing, and what people are doing in the classroom today, is shredding that DNA using things like inscription enzymes that can make DNA into more controllable parts in a way Repeat and then those pieces, depending on how big the pieces are, more or less migrate into the father gel from the bottom of the box from top to bottom.

A complete DNA molecule from a cell to a gel. It’s so big that it never enters the gel, so what scientists are doing, and what people are doing in the classroom today, is shredding that DNA using things like inscription enzymes that can make DNA into more controllable parts in a way Repeat and then those pieces, depending on how big the pieces are, more or less migrate into the father gel from the bottom of the box from top to bottom.

Types of electrophoresis

Conventional electrophoresis

Conventional electrophoresis is the traditional and most widely used clinical laboratory method for the separation of proteins and nucleic acids. This technique is usually performed on a rectangular slab gel, also called “area electrophoresis” because it can place multiple samples and controls on one gel and can be used to separate solutes in one run. It can also be used to isolate CSF and urine proteins, isoenzymes, lipoproteins and hemoglobin.

High resolution electrophoresis

High-resolution electrophoresis (HRE) is nothing more than conventional high-voltage electrophoresis. It is usually highly recommended if you clearly need higher protein (eg, isolation of CSF proteins for the diagnosis of multiple sclerosis, isolation of light chains in the urine for the early detection of multiple myeloma, etc.).

 Because the increase in voltage also increases the heat generated, the HRE includes a cooling device to prevent the proteins from denaturing and the gel and other components from drying out.  

Polyacrylamide (PAGE)

Acrylamide electrophoresis (also known as PAGE) is commonly used to isolate proteins based on molecular size and mass-to-mass ratio.

 With the help of vertical plates or gels embedded in vertical rods or cylinders, researchers can isolate DNA of 100 bp or less and analyze individual proteins in a single serum (such as genetic variants, isoenzymes).

 Apart from its simplicity and speed of separation, researchers like PAGE because gels are stable over a wide range of pH and temperature, and gels with different pore sizes can form.

Capillary electrophoresis (CE)

Capillary electrophoresis is performed on capillaries less than a millimeter in diameter (ie, very thin, fused silica capillary tubes with an internal diameter of 25 to 100 mm) and combines high-performance liquid electrophoresis and chromatography to facilitate analyte separation.

 Many researchers prefer to use CE because it requires only a small sample size, is very efficient, produces fast results, and can be easily automated. 

Isoelectric focus (IEF)

If you want to separate amphoteric compounds (such as proteins) more clearly, you must use this protocol. The IEF uses chemically injected gels to create a pH gradient on the gel surface and applies a very high voltage to facilitate the migration of protein molecules to a point where their net charge is zero (isoelectric point).

 Some of the advantages of using IEF are: ease of operation (ie it does not matter if the sample is used because the protein is always in the position according to its pl) and its high resolution. 

Immunofixation electrophoresis (IFE)

In general, IFE is used to diagnose monoclonal immunoglobulin gammopathies or monoclonal dilation of a single, dysfunctional antibody such as IgA, IgG, and IgM, the presence of which may indicate conditions such as multiple myeloma or Waldenstrom macroglobulinemia. It can also be used to study protein antigens and their broken down products.

Pulse field gel electrophoresis (PFGE)

You can not separate large DNA molecules over 50 kb (A kb) using AGE or PAGE in conventional electrophoresis systems, as the gel pore size is simply so small that it does not allow them to migrate.

 However, you can use pulse field gel electrophoresis (PFGE) to facilitate the successful division of large DNA molecules (up to 10 MB). 

PFGE effectively separates DNA fragments by applying a continuously changing electric current to the gel matrix.

 By replacing the positive and negative electrodes in the cycles during electrophoresis, the DNA molecules are forced to change direction and eventually break down into smaller pieces. 

PFGE is commonly used in genotype or genetic fingerprinting and is considered the gold standard in the subgroup of bacteria due to its simplicity and reproducibility. 

However, this protocol is very time consuming and requires a high level of skill. In addition, interpreting the results may be difficult because fragments are separated by their size (ie, segregation is not by sequence) and pieces of the same size may not come from the same chromosome.

Two-dimensional electrophoresis

In two-dimensional electrophoresis, the sample is separated using two separate separation techniques (eg IEF followed by PAGE or AGE) and identified in two dimensions with perpendicular angles.

 As the resulting bands are more dissolved by the second electrophoresis, you are more likely to get more information from your sample. 
Two-dimensional electrophoresis is highly specialized and is commonly used in proteomics and genetics research. While it can analyze thousands of proteins in one run, the technique requires a significant amount of prototype, limited reproducibility, and low throughput. 

In addition, this method only works with medium to large biomolecules and does not provide accurate measurements.

Gel and DNA electrophoresis

  • Electrophoresis enables you to detect pieces of DNA of different lengths.
  • DNA has a negative charge, so when an electric current is applied to the gel, the DNA migrates to the positively charged electrode.
  • Shorter strands of DNA move faster than longer strands in the gel, so the pieces are arranged in order of size.
  • Use paint,  fluorescent  Tags or  radioactive  The labels enable the DNA on the gel to be seen after separation. They appear as streaks on the gel.
  • A DNA marker with fragments of specified lengths usually passes through the gel at the same time as the samples.
  • By comparing the bands of DNA samples with DNA marker strips, you can determine the approximate length of the DNA fragments in the samples.

Application of electrophoresis

How to prepare gel

  • Agarose gel  Commonly used to visualize DNA fragments. The concentration of agarose used to make the gel depends on the size of the DNA fragments you are working with.
  • The higher the concentration of agarose, the denser the matrix, and vice versa. Smaller pieces of DNA are separated at higher concentrations of agarose, while larger molecules require lower concentrations of agarose.
  • To make a gel, agarose powder is mixed with an electrophoresis buffer and heated to high temperature to melt all the agarose powder.
  • The molten gel is then poured into a gel casting tray and a “comb” is placed at one end to pipette the sample wells.
  • When the gel has cooled and solidified (it now becomes opaque instead of transparent), the comb is removed.
  • Many people now use pre-made gels.
  • The gel is then placed in an electrophoresis tank and the electrophoresis buffer is poured into the tank to cover the gel surface. The buffer conducts electricity. The type of buffer used depends on the approximate size of the DNA fragments in the sample.

Preparation of DNA for electrophoresis

  • Prior to electrophoresis, a dye is added to the DNA sample to increase the viscosity of the sample, which prevents it from floating in the wells, so that the migration of the sample is visible through the gel.
  • A DNA marker (also known as the DNA size standard or ladder) is loaded into the first gel well. The parts in the indicator have a certain length, so they can be used to help approximate the size of the parts in the samples.
  • The prepared DNA samples are then pipetted into the remaining wells of the gel.
  • Once this is done, the cap is placed on the electrophoresis tank and we make sure that the direction of the gel and the positive and negative electrodes are correct (we want the DNA to migrate from the gel to the positive end).

Disassemble the parts

  • The electric current is then turned on to move the negatively charged DNA through the gel to the positive side of the gel.
  • Shorter lengths of DNA move faster than longer lengths, so move more when running a stream.
  • The distance that DNA has migrated in the gel can be visually assessed by monitoring the color buffer migration.
  • The electric current remains bright enough to ensure that the DNA fragments move long enough in the gel to separate them, but not long enough to leave the end of the gel.

الکتروفورز

sorce :wiki/Electrophoresis

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