Principles of electrophoresis technique used in the laboratory that results in the separation of charged molecules cations vs anions
By Live Dr - Thu Jan 15, 10:21 am
Electrophoresis is a technique used in the laboratory that results in the separation of charged molecules. Of the many physicochemical techniques, it occupy a position of primary importance. Amino acids, nucleotides, polypeptides, protein, and other compounds in a colloidal state can be separated by the way of electrophoresis.
Electrophoresis is defined as the migration of charged ions in an electric field. In metal conductors, electric current is carried by the movement of electrons, largely along the surface of the metal. In solutions, the electric current flows between electrodes and is carried by ions. The ions that migrate towards the anode, because of their anodic migration, are called “anions”. The ions which will migrate to the cathode are called “cations”.
There is a potential difference (voltage) between the anode and the cathode and if the solution between these is of constant composition and constant cross-section, the voltage gradient between them (dV/dx) will be linear, with units of volts cm-1.
An ion placed in such an electric field will experience a force:
F = electrophoretic force
K = a constant
q = net charge on the protein (atomic charges/protein molecule)
This force will cause the protein to accelerate towards either the cathode or the anode, depending on the sign of its charge. Of course there are other forces such as frictional force when ion move in the electric field. The influence of them can not be understood easily by a formula, so we omit it.
Electrophoresis exploit the fact that different ions have different mobility in an electric field and so can be separated by this way. Now we know the basic principle of electrophoresis, next we will talk about the major influencing factors of it.
2. Influencing factors
Movement of proteins depends on various aspects. Within the gel the molecules must pass through as they are moving from one pole to another. The smaller molecules can weave in and out of the matrix of the gel with more ease, compared with larger molecules. As a general rule, the molecules move rapid if it has more net charge, has a shape of ball and shorter diameter.
1) The buffer pH
It will influence the direction and rapid of the protein migration.
Movement of proteins depends on various aspects; one of them is the charges on the proteins. Proteins are sequence of amino acids that can be ionized depend on their acid or basic character. The protein’s net electric charge is the sum of the electric charges found on the surface of the molecule as a function of the environment.
The rate of migration will depend on the strength of their net surface charges: The protein that carries more +ve charges will move towards the cathode at a faster rate. On the contrary, the protein that carries more -ve charges will move towards the anode at a faster rate. In this regard, proteins can be separated based on their electric charges.
Depending on the pH of the buffer, proteins in a sample will carry different charges. At the pI (isoelectric point) of a specific protein, the protein molecule carries no net charge and does not migrate in an electric field. At pH above the pI, the protein has a net negative charge and migrates towards the anode. At pH below the pI the protein obtains a net positive charge on its surface and migrates towards the cathode.
2) The buffer ionic strength
It influences the proportion of the current carried by the proteins
At low ionic strength the proteins will carry a relatively large proportion of the current and so will have a relatively fast migration. At high ionic strength, most of the current will be carried by the buffer ions and so the proteins will migrate relatively slowly. An analogy might be useful in visualizing this effect of ionic strength. Imagine a bank where there are two counters – one for deposits the anode) and one for withdrawals (= the cathode), with electrons being the money. The ions may be considered as customers waiting to be served at either counter, which one can visualize as being at opposite ends of the banking hall.
In electrophoresis, therefore, a low ionic strength is preferred as it increases the rate of migration of proteins. A low ionic strength is also preferred as it gives a lower heat generation. Assuming a constant voltage, if the ionic strength is increased, the electrical resistance decreases but the current will increase. A high ionic strength buffer will therefore lead to greater heat generation, and so a low ionic strength is preferred.
3) The voltage gradient
The rate of migration will depend on the voltage gradient: There are more voltage gradient in the electric field, protein will move towards the anode (or the cathode) at a faster rate.
Liquid’s relative move upon solid medium in an electric field is called electoosmosis. In applied electric field, electoosmosis distort the sample stream and limit the separation. For example, Paper electrophoresis has poor resolution because of electoosmosis. The surface of paper has -e, so the buffer has +e derived from hydrogen ions because of electrostatic induction.
And then +e drive buffer to cathode in electric field. These flows distort the electrophoretic migration of sample by causing a varying residence time. Thus, sample will move more or less than normal.
3. The sort of electrophoresis
1) Paper electrophoresis
One of the earliest forms of zone electrophoresis for the separation was paper electrophoresis. In this a strip of filter paper was used as a medium to support a thin layer of buffer. Since the paper served only to support the buffer, paper electrophoresis can be considered as a form of free electrophoresis (as opposed to electrophoresis in a sieving gel, which will be discussed later.)
2) Cellulose acetate membrane electrophoresis (CAM-E)
CAM-E has a rather open, porous structure and a negative charge which, besides causing marked electroendosmosis, can result in the binding of positively charged proteins.
3) Agarose gel electrophoresis
CAM, in turn, has largely been replaced by agarose as a support medium for free electrophoresis in medical diagnosis. Although agarose is a gel, it has a macroreticular structure and thus does not impede the electrophoretic migration of molecules. Proteins are thus not usually retarded but nucleic acids can be kDa separated on the basis of their size by gel sieving. As with all gels, water cannot flow through agarose. There is therefore no necessity to maintain the two buffer reservoirs at the same height, since the buffer is unable to siphon through an agarose gel. Agarose has other advantages: it can be obtained in a form with zero -electroendosmosis, it can be cast onto a sheet of flexible plastic Gel AE Bond and, after staining and destaining, it can be dried onto the Gel- Bond to provide a durable record which is easily filed.
Other gel electrophoresis involve disc electrophoresis, polyacrylamide gel electrophoresis (PAGE), Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Isoelectric focusing electrophoresis, 2-D Electrophoresis, and so on.
There are other kinds of electrophoresis, such as powder electrophoresis and silk thread electrophoresis. Most of them are out of date and rarely used.
Cellulose acetate membrane electrophoresis
1．The blood serum
Serum proteins are separated into albumin and globulins. In other words, total protein = albumin + globulin. Globulins are divided into alpha-1, alpha-2, beta, and gamma globulins.
It is placed on CAM-E and exposed to an electric current. The various proteins move on the paper (migrate) to form bands that show the proportion of each protein fraction.
- Total protein: 6.4 to 8.3 g/dL
- Albumin: 3.5 to 5.0 g/dL
- Alpha-1 globulin: 0.1 to 0.3 g/dL
- Alpha-2 globulin: 0.6 to 1.0 g/dL
- Beta globulin: 0.7 to 1.2 g/dL
- Gamma globulin: 0.7 to 1.6 g/dL
2. Cellulose acetate membrane electrophoresis (CAM-E)
Cellulose acetate membranes give sharper protein bands and better resolution than paper, bind proteins less and have less endosmosis. As a result, CAM-E replaced paper electrophoresis and remained in use mainly in medical diagnostic laboratories for many years. The apparatus required for CAM-E is the same as for paper electrophoresis. Cellulose acetate membranes are white and opaque.
Take out a piece of cellulose acetate membrane by nipper. Be care to operate by the two ends, not middle, to avoid destroying the lane. Distinguish the smooth with rough surface, and marked in the rough surface by pencil. Be remember, samples must be loaded on the rough surface. And then put cellulose acetate membrane (CAM) into buffers at least 20 minutes.
Pick out CAM from buffer and put it on filter paper to absorb superfluous lipid. And then load the prepared sample. This is sample applicator, dip it into blood serum and then spot it on CAM mildly. Be attention to spot near one end, about 2.5 cm from end.
The experimental set-up for CAM electrophoresis is shown below. A strip of CAM, typically 2 x 10 cm. The strip is soaked in buffer, blotted briefly and suspended between supports in the apparatus. Buffer is added to both the anode and the cathode compartments: it is important that the levels in the two compartments are the same to prevent siphoning through the filter paper. The CAM is connected to the buffer by wicks, which must be the same width as the filter paper strip.
4) Staining: After electrophoresis, take out the CAM strip from buffer, dye it with the Sudan black B (one of dye) for 10 minutes.
5) Destaining：7% acetic acid is used to rinse CAM trip up to showing clear bands.
Agarose gel electrophoresis
1. Serum lipoprotein:
Dye the serum lipoprotein with the Sudan black B in advance, and add it in the agarose gel electrophoresis to separate every constituent of the serum lipoprotein clearly.
There are four kinds of lipoproteins in blood serum–chylomicra (CM), preβ-lipoprotein(very low density lipoprotein, VLDL), β-lipoprotein(low density lipoprotein, LDL), α- lipoprotein(high density lipoprotein, HDL). But only three of them can be distinguished after fasting diet for 12 hours or collecting serum on an empty stomach in the morning. They are LDL, VLDL, and HDL from cathode to anode after electrophoresis.
2. Agarose gel electrophoresis
Agarose is a polysaccharide obtained from seaweed. High grade agarose is expensive. Guess how much this 500 gram bottle costs. Would you believe $200?
You may be wondering what exactly a gel is, and what it has to do with agarose. Let’s find out by “making” a gel. Purified agarose is in powdered form, and is insoluble in water (or buffer) at room temperature. But it dissolves in boiling water. When it starts to cool, it undergoes what is known as polymerization.
Rather than staying dissolved in the water or coming out of solution, the sugar polymers crosslink with each other, causing the solution to “gel” into a semi-solid matrix much like “Jello” only more firm. The more agarose is dissolved in the boiling water, the firmer the gel will be. While the solution is still hot, we pour it into a mold so it will assume the shape we want.
Imagine you are a lipoprotein molecule. If you were inside an agarose gel, your environment would resemble a very dense 3-D spider web. If you are a small fragment, you could easily crawl through the spaces in between the webs. But as you increase in length, it gets harder and harder for you to fit through the spaces. If it were a race between you and another molecule, who would win? Do you think the same would hold true for any charged molecule?
1) Staining serum in advance（already prepared）
Dye the serum samples with the Sudan black B in advance, 10:1 mixed in 37oC for 30 minutes, and then centrifuged in 2000r/r.p.m for 5 minutes.
2) Prepare agarose gel
with water in concentration of 0.45％, and then put it in bath of boiling water. After melting, pour it on a piece of glass slide with haustorial tube, about 3 ml for everyone. Standing for about 30 min until the agarose solution become semi-solid matrix.
Make a slot in agarose matrix with special tool, locate at 2 cm from one end. Absorbing the liquid under the slot, load 15ul serum sample.
After loading, put gel glass slide to electrophoresis chamber just like CAM electrophoresis. Put through electricity，adjust 110～130 voltage，after 50 minutes, you will find separated bands.