Carrier amphoteric electrolyte pH gradient isoelectric focusing experiment
Carrier amphoteric electrolyte pH gradient isoelectric focusing experiment
Isoelectrofocusing, abbreviated as IEF or EF, was established in the 1960s as a means of separating and analyzing proteins, and has evolved rapidly over the past 30 years to become the highest-resolution electrophoresis technique available today. This experiment is from "Experimental Techniques of Protein Electrophoresis", edited by Guo Yaojun.
Operation method
Thin-layer analysis by isoelectric focusing
Principle
Proteins are separated and analyzed in a stable, continuous, linear pH gradient using differences in the isoelectric points of protein molecules or other amphipathic molecules. Therefore, the use of isoelectric focusing techniques for analysis is limited to proteins and amphiphilic molecules. The conditions for analysis are a stable, continuous and linear pH gradient in the gel.
Materials and Instruments
Acrylamide Monomer Reservoir Ammonium Persulfate Reservoir Move I. Isoelectric focusing of polyacrylamide gels For more product details, please visit Aladdin Scientific website.
Syringe Water bath
1. Gel production
(1) Preparation of gelation mold
The use of commercial glue is convenient and rapid, repeatability or good, but expensive, so the general laboratory are their own glue. Currently there are three methods of making glue: cover method, panning method and capillary filling method.
Cover plate method: cover plate method is simple, just like the production of microscope observation with the same sample. Pour a certain amount of glue on the slide and cover it with a coverslip. So it is easy to operate, but the reproducibility is very poor and it is not suitable for making large size gels. Molds can be made in the laboratory itself.
Translational method: The original Swedish LKB Ultromould can be used for translational gelation, the use of this mold is the most convenient operation, and also very easy to succeed, but the mold is expensive, and the size and thickness of the gel are limited by the mold.
Capillary filling method: compared with the first two methods, not only the mold is simple, and the gel thickness, size can be controlled by their own mold, filling method is also very easy to master. The author's laboratory are now using their own mold. Capillary filling mold consists of two clamps and two glass plates. The slightly shorter glass plate has an edge of a certain thickness on both sides. The size of the glass plates depends on the size of the gel to be made.
Pour a little hydrophobic silane on the clean, short glass plate with sides and spread it evenly on the glass plate with soft paper or cotton. Place a piece of plastic support film on the long, non-rimmed glass plate so that the film is close to the glass. Alternatively, use 1 mm thick flat glass or dampened cellophane. Cover the short glass plate with edges and clamp the glass plate on the edged side with clamps. The assembled mold is placed horizontally, ready for filling. 
(2) Solution Preparation
Acrylamide monomer storage solution: Dissolve 14.55 g of acrylamide and 0.45 g of N,N'-methoxybisacrylamide in 35 ml of distilled water, stir until dissolved, add 50 ml of distilled water and filter the solution, which can be stored for two weeks at 4℃.
10% (W/V) Ammonium Persulfate Reservoir Solution: Dissolve 0.1 g of ammonium persulfate in 1 ml of distilled water, solution must be freshly prepared. 
(3) Filling and removing glue
Use a syringe with a rubber tube to draw up the solution in the filter bottle and inject it evenly and slowly from one end of the mold between the glass plates of the mold. To prevent the gel solution from spilling on the lab bench, the mold can also be placed in a dish. 
Gel polymerization takes about 40 minutes to 1 hour. After polymerization, the gel can be removed when the refraction of light can be seen on the banded side of the mold. Insert a thin knife between the bottom glass plate and the plastic support film and gently pry the bottom glass and plastic support film apart. Carefully use the thin knife to peel the gel from the glass plate first along the edges of the glass plate, and then peel the whole gel with the support film. The polymerized gel can be used immediately or stored in a refrigerator at 4°C with some humidity. 
2. Isoelectric Focusing
Turn on the circulating water bath and set the cooling temperature. Generally 4~15℃, depending on the room temperature. When the room temperature is low, use a lower cooling temperature. On the contrary, when the room temperature is high or there is urea in the system, use a higher cooling temperature. Spread the prepared polyacrylamide gel on the cooling plate, coated with liquid paraffin or kerosene and avoid air bubbles to ensure good contact between the gel plate and the cooling plate. 
Wet the filter paper electrode strip with suitable electrode solution. Anodic and cathodic electrode solutions for different pH ranges can be selected according to Table 6.3. Place anodic and cathodic filter paper strips on each side of the gel. Some instruments use a coarse electrode in direct contact with the gel and do not require filter paper electrode strips.
Samples are added to the gel according to the grid on the cooling plate and the pre-designed experimental plan. Trace samples ( < 3 μl) can be added directly to the gel, dilute samples are added to the spiking filter paper, and isoelectric point standards are added on both sides or in the center. According to the principle of isoelectric focusing, the sample can be added at any suitable position on the gel, and the concentration is usually 0.5~2 μg/μl.
Place the electrodes in the center of the filter paper electrode strip respectively, and then connect the anode and cathode to the positive and negative poles of the power supply respectively. Turn on the power supply. Refer to Table 6.4 for electrical parameters. take care to ensure good contact between the electrodes and electrode strips. After half an hour of electrophoresis, remove the sample filter paper. For samples that are easily denatured, pre-focus for 15~30 minutes before adding samples. 
3. pH Gradient Measurement
There are three methods for pH gradient determination.
(1) An early method of measuring the pH gradient, especially when using disc electrophoresis, is to soak the gel in distilled water (or 10 mmol/L potassium chloride) cut into segments and then measure the pH of the soaking solution.
(2) After focusing, the pH can be measured with a surface electrode, and the value is measured every 1 cm from the cathode to the anode according to the grid on the cooling plate. To prevent spreading of the focusing band, refocus for 5-10 minutes after pH measurement. The pH gradient is then plotted on a gel scanner, camera system, or coordinate paper.
(3) If an isoelectric point protein standard is used, draw a pH gradient curve based on the position of the electrophoretic bands and the isoelectric point data after staining.
4. Fixation, staining, decolorization
After electrophoresis, discard the electrode strips and immediately fix the gel in fixative for 30 minutes. Discard the fixing solution and wash the gel with decolorizing solution for 5 minutes. Discard the decolorizing solution and put the gel into the staining solution at 60℃ for 10 minutes. Discard the staining solution, wash the gel with the decolorizing solution, and gently rub the surface of the gel with wet cotton. Replace the decolorizing solution several times until the blue color is completely removed from the background of the gel. The solution formulations are shown in Table 6.5. This is a quick method of staining with Thomas Brilliant Blue R-250. Depending on the nature of the sample and the requirements, different staining methods should be used. The methods described in "Conventional Polypropylene Diamine Gel Electrophoresis" and even some of the methods described in "SDS Polypropylene Coolamine Gel Electrophoresis" are suitable for isoelectric focusing electrophoresis. However, these methods in isoelectric focusing electrophoresis, due to the carrier of amphoteric electrolyte, decolorization is more difficult, and the sensitivity is not as good as conventional polyacrylamide gel electrophoresis and SDS electrophoresis, such as silver dyeing method in SDS electrophoresis sensitivity of up to 0.3 ng, but can only be detected in the isoelectric focusing 1~5 ng. 
5. Preservation
After decolorization, put the gel in the preservation solution for half an hour, take it out and dry it on a glass plate, wrap the gel plate with a piece of cellophane wetted with decolorizing solution, or wait for the gel to be sticky, press a piece of transparent plastic film, and let it dry, so that a piece of gel plate with stained protein bands can be preserved permanently.
Agarose gel isoelectric focusing
Agarose gel isoelectric focusing has the advantages of non-toxicity, short electrophoresis time, easy gel making, dyeing, decolorization and preservation, and can separate and analyze the compounds with molecular mass up to one million, which has a unique position in the separation and analysis of large molecular mass molecules.
1. Gel preparation
(1) Mold preparation
Agarose gels can be filled using the same molds as the polyacrylamide gel capillary filling method, but with a supporting film that adheres well to the agarose. The assembled molds are held in an oven at 70°C for 15 minutes.
(2) Preparation of agarose solution
Add 0.15 g of electroless endosmotic agarose to a triangular flask containing 14.1 ml of distilled water and bring to a boil on a water bath while stirring. About 10 minutes after the agarose has dissolved, stop heating and reduce the temperature of the water bath to 75°C. Add 0.9 ml of the desired agarose. Add 0.9 ml of carrier amphoteric electrolyte of the desired pH range to the agarose solution and mix well. Remove the mold from the oven. Use a syringe with a rubber tube to draw up the solution and fill between the two glass plates. While filling, elevate one end of the mold at an angle to allow the solution to flow in, and place the mold in a horizontal position immediately after filling. 
(3) Picking up the glue
Leave the mold at room temperature for at least 20 minutes after filling. Use a scalpel to cut off the excess agarose gel at both ends. Then hold the top glass plate and support film in one hand and the bottom glass plate in the other hand and break it, see Figure 6.15. After the agarose gel has solidified, leave it in the refrigerator for at least 1 hour to allow it to fully solidify. It can be stored at 4°C for up to one week while maintaining a certain temperature. Gently remove water from the surface of the gel with filter paper before use. Otherwise, the gel will be burned in a short circuit during electrophoresis. 
2. Isoelectric focusing
Isoelectric focusing is performed in the same way as for polyacrylamide gels. See Tables 6.6 and 6.7 for electrode solution and electrical parameters, respectively. 
3. pH gradient determination
The pH gradient is determined by the same method as for polyacrylamide gels, but since there is no protein standard for isoelectric focusing of agarose gels, surface electrodes are usually used.
4. fixation, staining, decolorization, preservation
After focusing electrophoresis, discard the electrode strip and immediately fix the gel in fixative for 10 minutes. Discard the fixative and wash the gel with 95% ethanol for 10 minutes. Blow the gel dry with hot air.
Place the gel in staining solution for 5 minutes. Stained gels are decolorized with a decolorizing solution. Gently rub the surface of the gel with cotton and replace the decolorizing solution until the blue color is completely removed from the background of the gel. Blow-dry with a hot air dryer for permanent storage.
The above is the commonly used method of staining the agarose gel with Caullemont Blue R250, but different staining methods may be used for different samples and requirements. Due to the large pore size of the agarose gel, decolorization is relatively easy, as shown in Table 6.8. 