Electrophoresis, Gel and cellulose electrophoresis protocol

Principle factors affecting, gel preparation and process of running. results interpretation, isoelectric focusing and capillary electrophoresis

1. Electrophoresis By Muhammad Asif Shaheen Lecturer, Department of Pathology, KEMU Lahore.

2. Electrophoresis is the migration of charged solutes or particles in an electrical field . Iontophoresis refers to the migration of small ions, whereas zone electrophoresis is the migration of charged macromolecules in a porous support medium such as paper, cellulose acetate, or agarose gel film. An electrophoretogram is the result of zone electrophoresis and consists of sharply separated zones of a macromolecule. In a clinical laboratory, the macromolecules of interest are proteins in serum, urine, cerebrospinal fluid (CSF), and other biologic body fluids and erythrocytes and tissue.

3. zone electrophoresis A ny  electrophoretic technique in which components are  separated into   zones or bands  in a buffer, and stabilized in solid, porous, or any other  supportmedium – eg , filter paper, agar gel, or  polyacrylamidegel T he   isoelectric point  ( pI ,  pH(I) ,  IEP ), is the  pH  at which a particular   molecule  carries no net  electrical charge  in the  statistical mean . The standard nomenclature to represent the isoelectric point is pH(I), [1]  although pI is also commonly seen Isoelectric focusing (IEF) is an electrophoresis technique that separates proteins based on their isoelectric point ( pI ). 

4. Electrophoresis consists of five components: The driving force (electrical power), The support medium , The buffer , The sample, and The detecting system .

5. Power Supply Power supplies operating at either constant current or constant voltage are available commercially. In electrophoresis, heat is produced when current flows through a medium that has resistance, resulting in an increase in thermal agitation of the dissolved solute (ions) and leading to a decrease in resistance and an increase in current. The increase leads to increases in heat and evaporation of water from the buffer , increasing the ionic concentration of the buffer and subsequent further increases in the current. The migration rate can be kept constant by using a power supply with constant current. This is true because, as electrophoresis progresses, a decrease in resistance as a result of heat produced also decreases the voltage.

6. Buffers Two buffer properties that affect the charge of ampholytes are pH and ionic strength. The ions carry the applied electric current and allow the buffer to maintain constant pH during electrophoresis. An ampholyte is a molecule , such as protein, whose net charge can be either positive or negative. If the buffer is more acidic than the isoelectric point ( pI ) of the ampholyte , it binds H, becomes positively charged, and migrates toward the cathode . If the buffer is more basic than the pI , the ampholyte loses H, becomes negatively charged, and migrates toward the anode.

7. A particle without a net charge will not migrate, remaining at the point of application. Generally, the most widely used buffers are made of monovalent ions because their ionic strength and molality are equal.

8. Support Material Cellulose Acetate Cellulose is acetylated to form cellulose acetate by treating it with acetic anhydride. Cellulose acetate, a dry, brittle film composed of about 80% air space, is produced commercially. When the film is soaked in buffer, the air spaces fill with electrolyte and the film becomes pliable. After electrophoresis and staining, cellulose acetate can be made transparent for densitometer quantitation. The dried transparent film can be stored for long periods .

9. Polyacrylamide Gel Polyacrylamide gel electrophoresis involves separation of protein on the basis of charge and molecular size. Layers of gel with different pore sizes are used. The gel is prepared before electrophoresis in a tube-shaped electrophoresis cell. The small-pore separation gel is at the bottom, followed by a large-pore spacer gel and, finally, another large-pore gel containing the sample. Each layer of gel is allowed to form a gelatin before the next gel is poured over it.

10. Starch Gel Starch gel electrophoresis separates proteins on the basis of surface charge and molecular size, as does polyacrylamide gel . The procedure is not widely used because of technical difficulty in preparing the gel.

11. Agarose gel electrophoresis The term " gel " in this instance refers to the matrix used to contain, then separate the target molecules. In most cases, the gel is a  crosslinked polymer  whose composition and porosity is chosen based on the specific weight and composition of the target to be analyzed. The gel is usually composed of different concentrations of  agarose  and a  cross-linker , producing different sized mesh networks. When separating larger nucleic acids (greater than a few hundred  bases ), the preferred matrix is purified agarose. In both cases, the gel forms a solid, yet porous matrix

12. "Most agarose gels are made with between 0.7% (good separation or resolution of large 5–10kb DNA fragments) and 2% (good resolution for small 0.2–1kb fragments) agarose dissolved in electrophoresis buffer. Up to 3% can be used for separating very tiny fragments but a vertical polyacrylamide gel is more appropriate in this case. Low percentage gels are very weak and may break when you try to lift them. High percentage gels are often brittle and do not set evenly. 1% gels are common for many applications

13. Apparatus for gel electrophoresis

14. Protocol: Gel Electrophoresis Pouring a Standard 1% Agarose Gel: Measure 1 g of agarose. Note:  Agarose gels are commonly used in concentrations of 0.7% to 2% depending on the size of bands needed to be separated Mix agarose powder with 100 mL 1xTAE in a microwavable flask . Microwave for 1-3 min until the agarose is completely dissolved (but do not over boil the solution, as some of the buffer will evaporate and thus alter the final percentage of agarose in the gel. Many people prefer to microwave in pulses, swirling the flask occasionally as the solution heats up.)

15. Let agarose solution cool down to about 50°C (about when you can comfortably keep your hand on the flask), about 5 mins . (Optional)  Add ethidium bromide ( EtBr ) to a final concentration of approximately 0.2-0.5 μg /mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light . Pour the agarose into a gel tray with the well comb in place. Note:  Pour slowly to avoid bubbles which will disrupt the gel. Any bubbles can be pushed away from the well comb or towards the sides/edges of the gel with a pipette tip. Place newly poured gel at 4°C for 10-15 mins OR let sit at room temperature for 20-30 mins , until it has completely solidified.

16. Loading Samples and Running an Agarose Gel: Add loading buffer to each of your digest samples. Note:  Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and will also allows you to gauge how far the gel has run while you are running your gel; and 2) it contains a high percentage of glycerol, so it increases the density of your DNA sample causing it settle to the bottom of the gel well, instead of diffusing in the buffer. Once solidified, place the agarose gel into the gel box (electrophoresis unit). Fill gel box with 1xTAE (or TBE) until the gel is covered.

17. Carefully load a molecular weight ladder into the first lane of the gel . Carefully load your samples into the additional wells of the gel. Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel. Note:  Black is negative, red is positive. (The DNA is negatively charged and will run towards the positive electrode.)  Always Run to Red. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box. (Optional)  If you did not add EtBr to the gel and buffer, place the gel into a container filled with 100 mL of TAE running buffer and 5 μL of EtBr , place on a rocker for 20-30 mins , replace EtBr solution with water and destain for 5 mins . Using any device that has UV light, visualize your DNA fragments

18. Analyzing Your Gel: Using the DNA ladder in the first lane as a guide (the manufacturer's instruction will tell you the size of each band), you can interpret the bands that you get in your sample lanes to determine if the resulting DNA bands that you see are as expected or not.

19. Cellulose Acetate Electrophoresis Apparatus An electrophoresis chamber or tank consists of two compartments separated by a partition. Each compartment has an electrode made of an inert material such as platinum. Each side is filled with equal amount of a suitable buffer solution . A bridge across the top of the partition holds a membrane or gel with each end of it in contact with the buffer directly or through paper wicks . The only connection between the two compartments is through this membrane. Sample is applied on to the membrane. Electrical power source is attached to the tank, which has an indicator for polarity.

20. Current of prescribed voltage is applied. Molecules start migrating through the membrane to anode or cathode depending upon their net charge. After the prescribed time, current is switched off and the membrane or gel is removed from the tank . It is then treated with suitable fixative and is stained to make the separated bands visible.

21. Reagents 1. Cellulose acetate strips of suitable size 2. Barbitone buffer, p H 8.6 3. Fixative solution Ponceau S, 0.5% w/v in 5% trichloracetic acid . Other protein stains such as commassie brilliant blue (CBB) or amido black can also be used. 5. Acetic acid, 5% v/v in water as destaining solution . 6. Clearing solution is prepared by adding 15 ml glacial acetic acid in 85 ml methanol

22. A typical capillary electrophoresis system consists of a high-voltage power supply, a sample introduction system, a capillary tube, a detector and an output device. Some instruments include a temperature control device to ensure reproducible results. This is because the separation of the sample depends on the electrophoretic mobility and the viscosity of the solutions decreases as the column temperature rises. 3  Each side of the high voltage power supply is connected to an electrode. These electrodes help to induce an electric field to initiate the migration of the sample from the anode to the cathode through the capillary tube.

23. The capillary is made of fused silica and is sometimes coated with polyimide. 3  Each side of the capillary tube is dipped in a vial containing the electrode and an electrolytic solution, or aqueous buffer. Before the sample is introduced to the column, the capillary must be flushed with the desired buffer solution. There is usually a small window near the cathodic end of the capillary which allows UV-VIS light to pass through the analyte and measure the absorbance. A photomultiplier tube is also connected at the cathodic end of the capillary, which enables the construction of a mass spectrum, providing information about the mass to charge ratio of the ionic species.

24. Isoelectric focusing

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