Electrophoretic separation and identification of proteins on cellulose acetate membranes
Electrophoretic separation and identification of proteins on cellulose acetate membranes
The isoelectric points of various proteins in mixed protein samples are different, and the amount of electrostatic charge carried by various proteins is different in barbiturate buffer at pH 8.6. 6 barbiturate buffer, the amount of electrostatic charge carried by various proteins is different, coupled with the different sizes of protein molecules, the speed of movement in the electric field is also different, for example, serum or ovalbumin samples in the albumin isoelectric point is lower than that of other proteins, in the pH8.6, more negatively charged than other proteins, coupled with the smaller molecules of albumin, therefore moving faster than other proteins in the electric field. For example, the isoelectric point of albumin in serum or ovalbumin samples is lower than that of other proteins, and at pH 8.6, it is more negatively charged than that of other proteins. In this way, the proteins in the sample on the cellulose acetate membrane can form zones, can be separated and analyzed protein composition. The purpose of this experiment is to master the basic principles of cellulose acetate membrane electrophoresis and the spotting technique and the use of horizontal electrophoresis tank.
Operation method
electrophoresis
Principle
The isoelectric points of various proteins in mixed protein samples are different, and the amount of electrostatic charge carried by various proteins is different in barbiturate buffer at pH 8.6. 6 barbiturate buffer, the amount of electrostatic charge carried by various proteins is different, coupled with the different sizes of protein molecules, the speed of movement in the electric field is also different, for example, serum or ovalbumin samples in the albumin isoelectric point is lower than that of other proteins, in the pH8.6, more negatively charged than other proteins, coupled with the smaller molecules of albumin, therefore moving faster than other proteins in the electric field. For example, the isoelectric point of albumin in serum or ovalbumin samples is lower than that of other proteins, and at pH 8.6, it is more negatively charged than that of other proteins. In this way, the proteins in the sample can form a zone on the cellulose acetate membrane, and the protein composition can be separated and analyzed. In addition, as purified proteins after electrophoresis should be a zone, otherwise it is not purified. Cellulose acetate membrane is the hydroxyl group of cellulose acetylation and get, will be dissolved in organic solvents (acetone, chloroform, vinyl chloride, ethyl acetate, etc.) after smearing into a uniform film, then into the cellulose acetate membrane, which has a strong permeability, molecular movement of non-resistance, as a support for zone electrophoresis protein electrophoresis with simple and fast, less sample, wide range of applications, separation of clear, there is no adsorption phenomenon. Advantages Proteins bind specifically to Amino Black 10B, but not to cellulose acetate membrane. After dyeing for a period of time and decolorization, the parts of the membrane without bound proteins do not show color, and the parts with proteins show obvious color.
Materials and Instruments
Serum protein Protein samples Move 1. Membrane preparation: Cellulose acetate membrane was cut into 8 cm × 2 cm strips (or the size of the membrane was determined according to the need), a line was drawn lightly with a pencil at 1.5 cm from one end of the membrane (spotting point), and immersed in barbiturate buffer until completely wet (about 20 min). Draw a line with a pencil 1.5 cm from one end of the membrane (at the point of sampling) and immerse it in barbiturate buffer until it is completely wet (about 20 min). Remove the soaked film with tweezers and place it between two layers of filter paper, pressing gently to remove excess liquid. 2. For more product details, please visit Aladdin Scientific website.
Barbiturates Distilled water HCl Amino black Sulfosalicylic acid Glacial acetic acid Acetic acid Anhydrous ethanol
Electrophoresis apparatus Electrophoresis tank Petri dishes Hematocrit pipette Pencil Ruler Slide Filter paper Tweezers
2. Spotting: Use a sharp heme pipette or other spotting tube to draw 2-3 μl of sample onto one section of the slide (the width of the slide should be smaller than the width of the membrane), and then gently touch the section of the slide with the sample to the spotting area of the membrane, and then the sample will be "imprinted" on the membrane in a line, so as to make the sample as narrow and homogeneous as possible.
3. Electrophoresis: The film with the sample spotted is set up on the electrophoresis tank with a filter paper bridge, so that the spotting end is close to the cathode, and the film is gently flattened with tweezers and cannot be attached to the bottom of the tank. Cover the tank lid, energize, control the current strength of 0.7 ~ 0.7 ~ 0.7 Control the current strength at 0.7 to 0.8 mA/cm of membrane width. Control the current strength at 0.7～0.8 mA/cm membrane width, when the albumin (yellowish color can be seen under careful observation) moves about 3 cm, then cut off the power supply to stop electrophoresis, generally for 40～60 min. 4.
4. Staining and rinsing: Remove the membrane and immediately immerse it in staining solution for 10 min, remove it and transfer it to 2.5% acetic acid for rinsing. Remove the membrane and put it into 2.5% acetic acid for rinsing and decolorization, and change the rinsing solution every 5 min until the background of the membrane is colorless (about 3 times), then a clear electrophoretic pattern can be observed.
5. Transparent treatment: After the rinsed electrophoresis pattern is completely dry (can be dried by hair dryer), immerse it in transparent liquid for 0.5 min and remove it immediately. After immersed in the transparent solution for 0.5 min, take it out immediately and stick it on the glass plate, after drying completely, it will become a transparent film, which can be scanned or photographed. If the glass plate is immersed in water, the film will be removed and dried out, and can be stored for a long time. 