Experiments on the determination of the purity of barley-wheat seeds by polyacrylamide gel electrophoresis
Experiments on the determination of the purity of barley-wheat seeds by polyacrylamide gel electrophoresis
In this experiment, polyacrylamide gel electrophoresis is used to extract and separate proteins in seeds, and the authenticity and purity of varieties can be identified according to the difference between the protein bands of different varieties and the standard bands. The purpose of this experiment is to master the principle of polyacrylamide electrophoresis, experimental methods and the application of this method in seed identification.
Operation method
Experiments on the determination of the purity of barley-wheat seeds by polyacrylamide gel electrophoresis
Principle
Alcohol soluble proteins extracted from seeds are well separated by the molecular sieving effect of the gel and the charge effect of electrophoretic separation, which reveals the type of protein bands by color development. Different varieties differ in the types of proteins contained within the seeds due to their genetic composition, and such differences can be identified using electrophoretic profiles, thus providing identification of variety authenticity and purity. Move I. Instruments and reagents: For more product details, please visit Aladdin Scientific website.
1, Instruments: electrophoresis apparatus (to meet the stabilized voltage 500V), centrifuge, vertical plate electrophoresis tank, clamp, 5ml, 10ml pipette, micro-sampler, polypropylene centrifuge tube.
2, reagents: urea, ethanol, glycine, methyl green, trichloroacetic acid, glacial acetic acid, hydrogen peroxide, ferrous sulfate, ascorbic acid, α-chloroethanol.
3, Pharmaceutical preparation:
(1) Protein extraction solution:
Wheat: 0.05 g of methyl green dissolved in 25 ml of α-chloroethanol, add distilled water to 100 ml. Keep at low temperature.
Barley: 0.05 g of methyl green dissolved in 20 ml of α-chloroethanol, add 118 g of urea, then add 1 ml of β-mercaptoethanol, add distilled water to 100 ml. Store at low temperature.
(2) Electrophoresis buffer: 0.4 g of glycine was dissolved in distilled water, 4 ml of glacial acetic acid was added, and the volume was fixed to 1000 ml. Store at low temperature.
(3) Gel buffer: 1.0 g of glycine was dissolved in evaporated water, 20 ml of ice acetic acid was added, and the volume was fixed to 1000 ml. Store at low temperature.
(4) 0.6% hydrogen peroxide: 2 ml of 30% hydrogen peroxide, add distilled water to 100 ml. Store at low temperature.
(5) Staining solution: 0.25 g of Caumas Brilliant Blue dissolved in 25 ml of anhydrous ethanol, add 50 g of trichloroacetic acid and add water to 500 ml.
(6) Gel solution: 20 g of acrylamide, 0.8 g of methylene fork bisacrylamide, 12 g of urea, 0.01 g of ferrous sulfate, 0.2 g of ascorbic acid, dissolve with gel buffer and set to 200 ml. Store at low temperature.
II.Experimental steps:
1, sample extraction:
General determination of each sample of 100 seeds, if a more accurate estimate of the purity of varieties, more seeds are required. If the results of the analysis to be compared with a standard value of purity, can be used in passing determination method (sepuential testing) to determine, that is, 50 seeds as a group, the necessary moment to determine the successive groups in order to reduce the workload. If only authenticity is to be identified, 50 seeds can be used.
Take wheat or barley seeds, crush them one by one with tongs (it is better to put a small piece of clean paper on the seeds when clamping them, so as to facilitate the cleaning of the tongs and prevent the contamination between the samples), put them in 1.5 ml centrifugal tubes, add the protein extraction solution (0.2 ml for wheat, 0.3 ml for barley) and mix them with full shaking, and then extract them for 24 hrs at room temperature, and centrifugate them for 15 mins under the condition of 18000 x g. The supernatant is used for electrophoresis, which is a good way of determining the authenticity of the samples. The supernatant was taken for electrophoresis.
2.Gel preparation:
Remove the gel solution and hydrogen peroxide solution from the refrigerator, aspirate 10 ml of gel solution, add 1 drop of 0.6% hydrogen peroxide, shake it well and pour it into the seal quickly, shake it slightly so that the whole slit is filled with gel solution, and let it polymerize and seal it in 5-10 minutes.
Aspirate 45 ml of gel solution, add 3 drops of 0.6% hydrogen peroxide, shake well and pour quickly between the gel plates, insert the sample comb immediately and let it polymerize in 5-10 minutes.
3.Sample feeding:
Carefully draw out the sample comb, add the glass plate on the electrophoresis tank, use filter paper or syringe to suck up the excess water in the sample tank, and then use a micro-sampler to suck up 10-20 μl of sample to add into the sample tank.
4, Electrophoresis:
Inject electrode liquid into the front and rear tanks, with the front tank connected to the positive pole and the rear tank connected to the negative pole. Then turn on the power supply and gradually increase the voltage to 500V electrophoresis, which is required to be carried out under the temperature of 15-20℃. The electrophoresis time is generally 60-80 minutes, the specific time can be estimated according to the migration time of methyl green, and the electrophoresis time is 2-2.5 times of the time required for methyl green to move to the front edge.
5, Staining:
Remove the adhesive plate carefully and stain it in the staining solution for 1-2 days. Generally there is no need to decolorize, but in order to make the band clear, it can be rinsed with water.
Third, identification:
The spectral band naming can be done by relative mobility method, or electrophoretic program method. According to the consistency of the composition and band type of the alcohol soluble protein spectral bands, and compared with the standard sample electrophoretic pattern, firm seed authenticity as well as determine the purity of varieties.