The phenomenon of a charged substance flowing in an electric field in the direction opposite to its polarity is called electrophoresis. Each protein in serum has a different isoelectric point, and when placed in a PH buffer with a higher isoelectric point than its isoelectric point, they will all be negatively charged and move toward the positive pole in the electric field. Since the isoelectric points and the charges and molecular weights of the proteins are different, they swim at different speeds in the electric field, and this feature is utilized to separate the proteins in the serum. This experiment is from Mudanjiang Medical College, undergraduate 5-year laboratory guide for testing majors.
Operation method
Serum protein cellulose acetate membrane electrophoresis detection test
Principle
The phenomenon of a charged substance flowing in an electric field in the direction opposite to its polarity is called electrophoresis. Each protein in serum has a different isoelectric point, and when placed in a PH buffer with a higher isoelectric point than its isoelectric point, they will all be negatively charged and move toward the positive pole in the electric field. Because the isoelectric point of each protein and the amount of charge and molecular weight are different, and therefore swim in the electric field at different speeds, the use of this feature to separate the various proteins in the serum.
Materials and Instruments
Cellulose acetate membrane Move Instruments: electrophoresis apparatus, electrophoresis tank, serum spiking tape, spectrophotometer, filter paper, scissors Caveat Reasons for failure:1. The electrophoretic pattern is not neat: uneven spotting, incomplete immersion of the film or high temperature resulting in partial drying of the film surface or water evaporation, deterioration of buffer: incorrect placement of the film during electrophoresis, so that the direction of the current is not parallel. 2. poor separation of protein fractions: too many samples, too low current, excessively dense film structure, poor water permeability, poor conductivity, etc. 3. light coloring in the middle of albumin after staining: due to insufficient staining time or old staining solution: if caused by high protein content, reduce the amount of serum or extend the staining time, generally to extend the time of 2 minutes is appropriate. If the time is too long, the percentage of globulin will increase and the A/G value will decrease. 4. Incomplete transparency of the film: the temperature does not reach 90 degrees above the specimen into the oven, the transparent liquid is old and insufficient soaking time. 5. Air bubbles on the transparent liquid: there is grease on the glass sheet, which makes the film partially detached or rolls poorly when applying the film. For more product details, please visit Aladdin Scientific website.
Electrophoresis apparatus Electrophoresis tank Serum spiking tape Spectrophotometer Filter paper Scissors
Experimental materials: cellulose acetate membrane Specification 2×8cm.
Experimental reagents:
l. Barbiturate-barbitone sodium buffer (PH8.6 + 0.1. Ionic strength 0.06): weigh 2.21g of barbiturate and 12.369 of barbitone sodium in 500ml of distilled water, heat to dissolve, and then replenish to less than 1L with distilled water after it is cooled to room temperature.
2. Amino black 10B staining solution: weigh the amino 10B 0.1g, dissolved in 20ml of anhydrous ethanol to dissolve. Take 2.5g of sulfosalicylic acid, dissolve it in 74.5ml of distilled water, then mix the two liquids and shake well.
3. Rinsing solution: 45ml of methanol, 5ml of glacial acetic acid and 50ml of distilled water, shake well.
4. 0.4 M sodium hydroxide solution.
Experimental operation:
1. Add the buffer to the electrophoresis tank and adjust the buffer in both sides of the tank so that they are at the same level.
2. Preparation of vinegar fiber membrane: take a piece of vinegar fiber membrane soaked in buffer, 1.5 cm from one end of the hairy side (negative side), use a pencil to draw a horizontal line, make a point sample marking, after numbering, place the vinegar fiber membrane in the barbiturate-barbitone sodium buffer for soaking, and remove it when fully saturated (generally twenty minutes) and clip it in the middle of a clean filter paper to suck off the excess buffer.
3. Straighten the hair side of the vinegar fiber membrane by attaching it upward to the support of the electrophoresis tank, and add 3-5 microliters of hemolysis-free serum at the horizontal line along the horizontal line by drawing it up with a micropipette. The sample should keep a certain distance from the edge of the membrane, so as not to distort the protein zone band in the electrophoresis pattern, after the serum penetrates into the membrane, reverse the vinegar fiber membrane, so that the optical surface is flat and straight on the support of the electrophoresis tank, and connect the ends of the membrane and buffer with double filter paper or four layers of gauze, and leave it for a few moments.
4. Turn on the power supply, pay attention to the positive and negative poles on the vinegar fiber membrane, do not connect the wrong one. Voltage 90-150 volts, current 0.4-0.6mA/cm width (different electrophoresis apparatus may require different voltage and current, should be flexible). Current may be different for different electrophoresis instruments, it should be grasped flexibly); turn on the power for 45 minutes in summer and 60 minutes in winter, and then turn off the power when the electrophoresis zone unfolds about 25-35mm.
5. Staining: After electrification, remove the film and immerse it directly in amino black 10B staining solution for 5-10 minutes (until the albumin is stained through), and then bleach off the remaining dye in the rinsing solution until the background is colorless.
6. Quantification: The rinsed film should be dried, and the stained protein zones should be cut off and put into the corresponding test tubes. In the albumin tube, add 6 ml of 0.4M sodium hydroxide solution (absorbance multiplied by 2 when calculating), and the rest of them should be added 3 ml each and shaken for several times, and then put into the water tank at 37 degrees Celsius for 20 minutes, so as to make its dyes leach out. For the leached amino black 10B, read the absorbance of each tube with a spectrophotometer at a wavelength of 600-620nm, and then calculate the respective content (cut a strip of the membrane of the same width as the albumin zone in the protein-free zone of the vinegar fiber membrane as a blank control).
Calculation:
Protein % of each component=Ax/At×100％
Fractional protein g/L = % fractional protein × total serum protein g/L
AX table absorbance of each component protein At table sum of absorbance of each component protein
Reference values: 