Serum protein polyacrylamide gel has the advantages of good mechanical strength, elasticity, transparency, high chemical stability, non-electroosmosis, simple equipment, low sample size and high resolution, and can be controlled by the monomer concentration or the ratio of monomer and crosslinking agent polymerization into gels with different pore sizes, used for the separation of proteins, nucleic acids and other substances, qualitative and quantitative analysis.
According to the shape of the gel, it is divided into disk electrophoresis and plate electrophoresis. Disc electrophoresis is named after the use of discontinuous buffer pH for electrophoresis in an upright glass tube, and at the same time, the sample mixture separates to form a narrow, disc-shaped band, so the name disc electrophoresis became a pun on discontinuous and disc-shaped. Discontinuous disc electrophoresis has a high resolving power due to the presence of three effects, the concentration effect, the charge effect and the molecular sieve effect.
Operation method
Polyacrylamide gel electrophoresis of serum proteins
Principle
Serum protein polyacrylamide gel has the advantages of good mechanical strength, elasticity, transparency, high chemical stability, no electro-osmosis, simple equipment, low sample volume and high resolution, and can be polymerized into gels with different pore sizes by controlling the concentration of monomers or the ratio of monomers and cross-linking agents, which is used for the separation, qualitative and quantitative analysis of proteins, nucleic acids and other substances. According to the shape of the gel, it is divided into disk electrophoresis and plate electrophoresis. Disc electrophoresis is named after the use of discontinuous buffer pH for electrophoresis in an upright glass tube, and at the same time, the sample mixture separates to form a narrow, disc-shaped band, so the name disc electrophoresis became a pun on discontinuous and disc-shaped. Discontinuous disc electrophoresis has a high resolving power due to the presence of three effects, the concentration effect, the charge effect, and the molecular sieve effect.1. Concentration effect: the tube is filled with three different gel layers, see accompanying figure, the upper layer is the sample gel, the second layer is the concentration gel, both of which are large pore gels, which are buffered with Tris-HCL buffer pH 6.7. The third layer is the separation gel, which is a small pore gel Tris-HCL buffer, pH 8.9. the upper and lower electrophoresis tanks were fully buffered with Tris-glycine buffer, pH 8.3. this resulted in discontinuities in gel pore size, pH, and buffer. Under these conditions, HCL is almost entirely ionized to Cl-, a very small fraction of the molecules of glycine dissociates to NH2CH2COO-, and generally acidic proteins can dissociate and become negatively charged. When the electrophoresis system is energized, these three kinds of negative ions move towards the positive pole. According to the size of the effective swimming rate, the fastest one is called the ion or the first ion (here Cl-), and the slowest one is called the slow ion or the subsequent ion (here NH2CH2COO-). When electrophoresis begins, the fast ion is in the lead, and a region of low ion concentration, the low conductance region, forms after it. Conductance is inversely proportional to the voltage gradient, so the low-conductance region gives rise to a higher voltage gradient. This high voltage gradient causes the proteins and slow ions to move faster behind the fast ions. In the fast ions and slow ions move at an equal rate after the establishment of a steady state, the fast ions and slow ions between the interface caused by a constant movement to the anode, because the effective mobility of protein is located exactly between the fast and slow ions, so the protein sample is sandwiched between the concentration of a narrow layer, this concentration effect can make the protein concentration of hundreds of times. 2. Charge effect: protein samples in the interface is concentrated into a narrow layer of highly concentrated protein. Charge effect: the protein sample at the interface is concentrated into a narrow region of high concentration of protein, but because each protein molecule carries a different effective charge, and therefore different mobility, so a variety of protein samples according to the mobility of the order of the order of the speed of a disk zone. In the separation gel when the charge effect still plays a role. 3. molecular sieve effect: when the concentrated protein samples from the concentrated gel into the separation gel, the pH and gel pore size suddenly change, choose the separation gel pH 8.9 (electrophoresis when the actual measurements are 9.5), so that close to the pka of glycine (9.7 ~ 9.8), so that the slow ions dissociation increases, and thus its mobility increases, this time, slow ions The slow ion mobility exceeds the effective mobility of all proteins, and the high voltage gradient no longer exists. At this time, the various proteins are not only different in molecular weight or configuration, but also different in friction when passing through the separation gel of a certain pore size under a homogeneous voltage gradient and pH condition, and different in the degree of blockage, and different in the performance of the mobility rate and are separated.
Materials and Instruments
Separation buffer Monomer crosslinker Concentration buffer Accelerator: catalyst Electrode buffer Amino black stain Acetic acid Sucrose Move I. Reagents For more product details, please visit Aladdin Scientific website.
1. Separation gel buffer (pH 8.9): take Tris 36.3 g and add 1NHC148 ml, then add water to 100 ml.
2. Monomer crosslinking agent: take acrylamide 30g, N-N-methylene bisacrylamide 0.8g, add distilled water to 100ml.
3. Concentrated gel buffer (pH 6.7): take Tris 5.98g plus 1NHC148ml, add distilled water to 100ml.
4. Accelerator: tetramethylethylenediamine.
5. Catalyst: 10% ammonium persulfate, take 1g of AP and add water to 10ml. prepare before use.
6. Electrode buffer (pH8.3): weigh Tris 6g, glycine 28.9g dissolved with distilled water to 100ml, diluted to 1000ml.
7.0.5% Amino Black Staining Solution: Take Kaomasland R-250 0.5g, 7% ice acetic acid 10ml, dissolve and add water to 100ml, mix well.
8.7% acetic acid: take 19ml of acetic acid with 37% content and add water to 100ml.
9.20% sucrose 100μl and 0.025% bromophenolan 5μl to form a sample dilution.
Preparation of gel column
1.Take 0.5×100cm glass tube, from the other end of the measurement of 7cm, 8cm at two places, respectively, with a glass pencil scribe line, the starting end of the tube mouth with a small piece of cellophane wrapped in a good, inserted into the rubber pad, vertical stand on the test tube rack.
2. Take 2 small beakers, labeled, first formulated according to the table separation gel, to be separated from the gel polymerization and then with the concentrated gel.
Pay attention to remove the air bubbles in the solution
3. Take the No. 1 cup after mixing the solution, with a burette suction to add the above glass tube to the scale (from the bottom of about 7cm) and then carefully with a glass tube in the gel on the cover with a 0.5cm thickness of the water layer, will be placed on the glass tube vertically on the test-tube rack for about half an hour or more, after the completion of the polymerization of the gel, with a filter paper suction covered with a layer of water, which is the separation of the gel. Prepare the concentrated gel according to the table, shake well, use a dropper to suck this liquid added to the above gel layer, to the second scale (8cm). The same was covered with a 0.5 cm thick water layer and placed vertically on the test tube rack. After polymerization, absorb the water layer with filter paper, which is the concentrated gel.
Third, add samples
Add 1/3 volume of 50% glycerol or 20% sucrose solution to the serum, add a small amount of 0.5% bromophenol blue as a tracing agent, mix well, and then add 30-50μ of this sample to the concentrated gel. Add electrode buffer along the wall of the tube until the end of the item.
Electrophoresis
1. Insert the prepared gel column into the small hole at the bottom of the electrophoresis tank, and the gel tube vertically in the upper and lower tanks, and mark it well. Add the electrode buffer (Tris-glycine) in the upper and lower tanks and insert the electrode with the upper negative and lower positive.
2. Adjust the current to 3mA/tube, voltage 100-200V, when the bromophenol blue tracer is close to the bottom of the tube (about 30 minutes to 1 hour of electrophoresis), cut off the power supply, and take out the gel tube (buffer is poured into the recovery cup).
V. Gel stripping
Remove the gel tube, use a syringe with a 10cm long needle containing distilled water as lubricant, insert the needle between the gel column and the wall of the tube, rotate the glass tube while injecting water until the gel column is separated from the wall of the tube, and gently pressurize it with a washing ball at one end so that the gel column slowly slides out of the glass tube.
VI. Staining and decolorization
Remove the gel column from the tube and immerse it in 0.5% to 1% amino black dye solution. Generally, the dyeing takes about 10 to 15 minutes, but some proteins need longer time. After dyeing, wash away the excess dye with water and put it into 7% acetic acid for decolorization until all the remaining black is removed. About 30 minutes.