Determination of isoelectric point of proteins by isoelectric focusing electrophoresis
Determination of isoelectric point of proteins by isoelectric focusing electrophoresis
Isoelectric focusing (IEF for short) is a new technique that appeared in the mid-sixties. In recent years, IEF has made new progress and has rapidly developed into a mature modern biochemical experimental technique. At present, IEF has been able to distinguish biological molecules with a difference of only 0.001pH in the isoelectric point (pI). Due to its high resolution, good reproducibility, large sample capacity, easy and rapid operation, it has been widely used in biochemistry, molecular biology and clinical medical research.
Operation method
Determination of isoelectric point of proteins by isoelectric focusing electrophoresis
Principle
Experimental PrincipleThe protein molecule is a typical amphipathic electrolyte molecule. It dissociates into negatively charged anions at pH greater than its isoelectric point and swims toward the positive end of the electric field, and into positively charged cations at pH less than its isoelectric point and swims toward the negative end of the electric field. This swimming can only stop at a pH equal to its isoelectric point, i.e., when the net charge of the protein is zero. If there is a pH gradient in the environment, a variety of different isoelectric points of the protein mixture of samples for electrophoresis, then under the action of the electric field, regardless of the original distribution of these protein molecules, a variety of protein molecules will be in accordance with the size of their respective isoelectric points in the pH gradient in the corresponding position to the position for focusing after a certain amount of time after electrophoresis, protein molecules with different isoelectric points will be focusing on different positions. After a certain period of electrophoresis, protein molecules with different isoelectric points will focus at different positions. According to the size of the isoelectric point, biomolecules in the pH gradient of a corresponding position to focus the behavior is called "isoelectric focusing". Isoelectric focusing is characterized by the use of a substance called amphoteric electrolyte carrier in the electric field constitutes a continuous pH gradient, so that proteins or other samples with amphoteric electrolyte properties to focus, so as to achieve the separation, determination and identification of the purpose. Amphoteric electrolyte carrier, in fact, is a mixture of many isomers and homologues, they are a series of polycarboxy polyamino aliphatic compounds, molecular weight between 300 ~ 1000. The commonly used imported amphoteric electrolytes are Ampholine and Pharmalyte produced by Pharmacia-LKB of Sweden, which are expensive. Domestic amphoteric electrolytes produced by the Institute of Radiation Medicine of the Chinese Academy of Military Medical Sciences and the Shanghai Institute of Biochemistry are cheap and of good quality. Amphoteric electrolytes can form a smooth and continuous pH gradient from the positive pole to the negative pole with a gradual increase in pH value under the action of a DC electric field. If the content of amphoteric electrolytes with different pH values is more uniformly distributed with respect to the pI value, then the pH gradient will be more linear. The requirements for Ampholine amphoteric electrolytes are high buffering capacity, good electrical conductivity, low molecular weight, and non-interference with the sample being analyzed. It is extremely important to keep the pH gradient stable during and after focusing when the applied electric field is removed. In order to prevent diffusion and stabilize the pH gradient, it is necessary to add an anti-convection and diffusion support medium, the most commonly used support medium is polyacrylamide gel. When the polyacrylamide gel isoelectric focusing electrophoresis, gel column that is the pH gradient, when the protein sample electrophoresis to a part of the gel column, and the pH value of this part is exactly equal to the isoelectric point of the protein, the protein that is the focusing of the formation of a zone, as long as the pH value of the part of this zone, that is, for its isoelectric point. The longer the electrophoresis time, the more concentrated and narrower the zone of protein focusing, thus improving the resolution. This is a major advantage of isoelectric focusing, unlike other electrophoresis, where the zone spreads when the electrophoresis time is too long. Therefore, isoelectric focusing electrophoresis can not only determine the isoelectric point, but also can separate and identify the mixed biomolecules with different isoelectric points. Early isoelectric focusing electrophoresis is vertical tube type, which is characterized by the system is closed, not in contact with air, can prevent the sample oxidation. In recent years, ultra-thin layer horizontal plate type isoelectric focusing electrophoresis has been developed. The advantage of this method is to add a large number of samples, saving the gender electrolyte, electrophoresis after fixation, staining, drying are very fast and easy, its biggest advantage is to prevent the electrode liquid electroosmosis caused by the positive and negative poles pH gradient drift. There are four methods to determine the pH gradient: 1. cut the strip into small pieces, soaked in water, use precision pH test paper or imported slender pH composite electrode to determine the pH value, and then make a graph; 2. use the surface pH microelectrode to directly determine the pH value of each part of the strip, and then make a graph; 3. use a set of proteins with different pI values as a set of known standards, and then determine the pH gradient of standard curves; 4. use the strip at -70 ℃ frozen and cut into 1.5 pieces, and then dry it. 4. Cut the strip into 1 mm slices after freezing at -70℃, add 0.5 ml of 0.01M KCl, and measure the pH with a microelectrode.
Materials and Instruments
Acrylamide Methacrylamide Amphoteric Electrolyte Ampholine Ammonium Persulfate TEMED Phosphoric Acid NaOH Trichloroacetic Acid Move Instruments and appliances Caveat Remarks1. Requirements for amphoteric electrolytesAmphoteric electrolyte is the key reagent for isoelectric focusing, so special attention should be paid to both the quality and quantity of amphoteric electrolyte. The amphoteric electrolyte content of 2% to 3% is suitable for the formation of a good pH gradient. Since the carrier amphoteric electrolyte is a mixture of polyethylene polyamine and acrylic acid in an addition reaction, it cannot be used because it will be separated into bacteria and decomposed for too long. 2. Instructed to pay attention to(1) Acrylamide is preferably recrystallized.(2) Ammonium persulfate must be newly prepared.(3) All water should be double-distilled water.(4) The glass plate and template must be placed horizontally when making glue.(5) The template should be straight and smooth, otherwise it is easy to splash when filling glue. 3， to prevent burning glue(1) Plate and other points of focusing electrophoresis glue is very thin (0.6 mm), when asked to willow in 8mA, the voltage can rise to more than 550V, due to cathode drift, resulting in local current is too large, the glue can not afford to be burned off.(2) The way to prevent burning glue: pay attention to observation, steady current 8mA, the voltage rises to 550V, immediately turn off the power. Use constant power electrophoresis instrument to control the output power in the specified range. Within a certain power range, improve the cooling conditions so that the heat generated by the current can be dissipated in time.(3) If the glue is burned, remedy the situation by replacing the burned-off position with a wide electrode strip pressed over the broken seam or by adding an electrode strip on the inside of the power supply electrode strip. 4. Precautions for mixing productionThe fixing solution can denature the protein and no longer spread, so it must be changed more than once; after electrophoresis to take the glue, fixing, staining until making dry glue plate must be careful to prevent the glue from being damaged. For more product details, please visit Aladdin Scientific website.
Electrophoresis apparatus Vertical tubular garden-plate electrophoresis tank set Syringes and needles Pipettes Small beakers Petri dishes Straightedge Knife Precision pH test paper and pH meter with slender compound pH electrodes Plastic film and rubber bands
1. Electrophoresis apparatus
2. One set of vertical tubular garden-plate electrophoresis tank
3. Syringe and needle
4. Pipette: 10 ml, 5 ml, 2 ml, 1 ml, 0.1 ml
5. a number of small beakers
6. a set of Petri dishes
7. ruler
8. knife
9. precision pH paper and pH meter with a thin composite pH electrode
10. plastic wrap and rubber bands
reagents
1. acrylamide
2. methylenedioxyacrylamide
3. Amphoteric electrolyte Ampholine (40%, pH 3.5-9.5)
4. Amine persulfate (catalyst)
5. TEMED (tetramethylethylenediamine) (accelerator)
6. Phosphoric acid
7. NaOH
8. trichloroacetic acid (TCA)
Solution Preparation
1. Acrylamide reservoir solution (30% acrylamide, 2.6% crosslinking): 30 g acrylamide and 0.8 g methylenebisacrylamide dissolved in H2O, volume fixed to 100 ml, filtered off insoluble matter and stored in a brown bottle at 4℃ for several months. (for class use) (another formulation is 29.1 g acrylamide and 0.9 g methylenebisacrylamide dissolved in H2O, fixed to 100 ml, crosslinking degree is 3.0%).
2. Amphoteric electrolyte Amphline (40%) is added at 50 ml/ml of gel.
3. Amine persulfate: Prepare at a concentration of 1 mg/ml on the same day, and dispense 100 ml for the entire shift. The amount to be added to the gel solution is 0.5 mg/ml of gel solution.
4. TEMED: Add 1ul/ml of gel to the gel.
5. protein samples: two proteins with large differences in isoelectric points are used, and the volume of each protein in each vertical tube is limited to <100ug. The protein samples are prepared to a concentration of 5 mg/ml each. Dispense 2.5 ml for class use.
6. Fixing solution: 10% trichloroacetic acid, 50 ml per group.
7. anode electrode solution: 0.1M H3PO4
3.4 ml concentrated phosphoric acid (85%) plus H2O to 500 ml, 500 ml for each electrophoresis bath.
8. cathode electrode solution: 0.5M NaOH
2 g NaOH plus H2O dissolved to 500 ml, 500 ml for each electrophoresis tank.
Procedure
1. Gel preparation 
Ammonium persulfate is the catalyst for the polymerization of the gel, so it is added last and shaken immediately after addition, because the gel will be polymerized quickly and must be filled immediately. Usually, the chemical polymerization of the glue needs to be depressurized and pumped before the addition of ammonium persulfate, but the omission of this pumping step does not affect the results of the experiment.
2. Tubing
Each student loads two tubes, four per assembly. Wash your hands with soap first, then wash the glass tubes in the disc electrophoresis tank, seal the bottom end with plastic film and rubber band, place them vertically on the test tube rack, and use a pipette to move the prepared gel into the tubes (the capacity of each glass tube is about 1.5~1.8 ml), and the liquid level was added to 1 mm from the mouth of the tubes, then gently add a small amount of H2O by syringe and carry out water sealing to eliminate the curved moon surface to make the top of the gel column flat. The tube was polymerized vertically for about 30 minutes, and the refractive surface under the water seal could be observed when the polymerization was completed.
3. Slotting and electrophoresis
Use a strip of filter paper to suck off the water seal at the upper end of the tube, remove the film at the lower end, with the water seal end upward, insert the tube vertically into the disc electrophoresis tank, adjust the height of each tube, and write down the tube number. Adjust the height of each tube and note down the tube number. About 1/3 of each tube should be in the upper tank and 2/3 in the lower tank. Add 500 ml of 0.1M H3PO4 to the upper tank and 500 ml of 0.1M NaOH to the lower tank, submerge the mouths of the tubes and the electrodes, and suck the air bubbles from the mouths of the tubes with a syringe or a dropper. Connect the upper tank to the positive pole and the lower tank to the negative pole, turn on the electrophoresis instrument, focus the electrophoresis for 2 to 3 hours at a constant voltage of 160V, and stop the electrophoresis when the current is close to zero and no longer decreases.
4. Gel stripping
Remove the tube, wash the tube and both ends twice with H2O, gently insert the needle along the wall of the tube with a syringe, and inject a little H2O into both ends of the tube while rotating the tube and inserting the needle, the glue strip will slide out by itself, and if it doesn't, it can be gently squeezed by the earwash ball. The strip is placed in a small petri dish, remember the positive end is the "head", the negative end is the "tail", if you can't tell the difference, use pH test paper to identify the acidic end is positive, the alkaline end is negative.
5. Fixation
Take 2 strips of adhesive tape in a small petri dish, pour 10% trichloroacetic acid solution until it does not exceed the adhesive tape, for fixation, about half an hour later, you can see the white precipitation of protein in the adhesive tape band. After fixation, pour out the fixing solution and use a ruler to measure the length of the strip "L2" and the length "L" from the positive end to the center of the white precipitation band of protein (i.e., the focusing part).
After fixation, the gel can be scanned with 280nm or 238nm wavelength on a Cortex 860 UV/Vis Spectrophotometer, and then the scanning graph can be used for corresponding measurements and calculations.
6. Determination of pH gradient
Place the unfixed gel strip in another petri dish and measure the length of the pH strip to be measured "L1" with a straightedge. In the order of positive to negative, cut the strip into 10 mm long pieces with tweezers and a knife, place them in small test tubes, add 1 ml of H2O, soak for more than half an hour or overnight, and then measure the pH value of the leachate in each tube with a carefully calibrated pH meter with a long and thin pH composite electrode.
Data processing
1. Use the length of the adhesive strip (mm) as the horizontal coordinate and the pH value as the vertical coordinate to make a graph, and obtain a pH gradient curve. The pH value of each tube is the average of the pH values of the 10 mm strips. The pH value is taken as the pH value at the center of the 10 mm segment, i.e., 5 mm.
2. Calculate the actual length, L, from the focusing site of the protein to the positive end of the strip using the formula below: 
L' - the length from the center of the white precipitation zone of the measured protein to the right end of the strip.
L1 - the length of the strip for pH measurement.
L2 - length of the strip after fixation
3. According to the calculated L, the corresponding pH value is found on the pH gradient curve, which is the isoelectric point of the protein.
4. Draw a schematic diagram of the strip after fixation.