[principle] The protein is disulfide bond-reduced, hydrogen-bonded, etc. in the molecule under the action of sodium dodecyl sulfate (SDS) and mercaptoethanol to form an SDS-protein polypeptide complex at a ratio of 1.4 g SDS/1 g protein, the complex Negatively charged, it can migrate to the positive electrode in polyacrylamide gel electrophoresis, and mainly due to the molecular sieve action of the gel, the migration rate is related to the molecular weight of the protein, so that the protein polypeptide can be concentrated and separated. Polyacrylamide gel electrophoresis separation of proteins mostly uses a discontinuous buffer system, which is mainly divided into lower concentration layered glue and higher concentration separation gel to prepare gel buffer, pH and ionic strength. Correspondingly, when electrophoresis, the SDS-polypeptide complex in the sample moves along the moving interface, forming a very thin layer on the surface of the separation gel, greatly concentrating the volume of the sample, ie SDS-polyacrylamide gel electrophoresis. Concentration effect. [Reagents] 1. 30% gel stock solution: Weigh 29 g of acrylamide (Acr) and 1 g of methylidene-bisacrylamide (Bis), add distilled water to 100 ml, and filter and store. 2, 10% SDS: Weigh 10 g of SDS and add distilled water to 100 ml. 3, 10% persulfate amine (AP) 4, N, N, N', N' tetramethylethylenediamine (TEMED) 5, 5 × electrophoresis buffer: 15.1 g of Tris, 94 g of glycine, 5 g of SDS were added to 900 ml of distilled water, and then mixed, and distilled water was added to 1 000 ml. 6. 1.5 mol/L Tris-HCl (pH 8.8) and 1.0 mol/L Tris-HCl (pH 6.8) 7, electrophoresis loading buffer 10mmol/L pH6.8 Tris 200mmol/LDTT 4% SDS 0.2% bromophenol blue 20% glycerin 8. n-butanol [equipment] 1, pipette 2, pipette 3, beaker 4, vertical electrophoresis tank 5, electrophoresis instrument [Steps] 1. Prepare separation gel concentration 8%, volume 15ml H2O 6.9ml 30% gel stock solution 4.0ml 1.5mol/L Tris-HCl (pH8.8) 3.8ml 10% SDS 0.15ml 10% persulfate amine 0.15ml TEMED 0.01ml 2. Once TEMED is added, carefully mix the separation gel into the gap of the prepared glass plate immediately after mixing to leave enough space for the layered glue. A few milliliters of n-butanol was gently added to the top layer with a capillary tube to prevent the inhibition of gel polymerization by oxygen in the air. 3. After the polymerization is completed, the covering liquid is poured off, and the upper portion of the gel is washed with deionized water several times, and the remaining liquid at the top is absorbed by the absorbent paper as much as possible. 4. Prepare 5 ml of 5% layered glue, insert the comb, carefully avoid mixing bubbles, and place it at room temperature vertically. H2O 3.4ml 30% gel stock solution 0.83ml 1.0 mol/Ltris-HCl (pH 6.8) 0.63 ml 10% SDS 0.05ml 10% persulfate amine 0.05ml TEMED 0.01ml 5. In the layering gel polymerization, mix the sample with the loading buffer and heat at 100 °C for 3 min. 6. After completion of the layering gel polymerization, the comb holes are rinsed with deionized water to remove unpolymerized acrylamide, and the gel is placed in an electrophoresis tank. 7. Sample loading 8. Electrophoresis: The initial voltage is 8V/cm gel. After the dye enters the separation gel, increase the voltage to 15V/cm gel and continue electrophoresis until the dye reaches the bottom of the separation gel and disconnect the power. 9. Remove the gel, fix, stain or perform a Western blot analysis. Another important function of amino acids is to provide energy to the human body. Normally, a healthy body on a general diet will use carbohydrates as its main fuel, but when the main source is depleted due to strenuous exercise, protein and amino acids can be used as a last resort. Amino acids also play an important role in food taste. Protein doesn't have much taste, but each amino acid has its own taste, and combining them is one of the important factors that define the taste of food Amino Acid,Asn Amino Acid,Leucine Powder,Food Grade L-Tryptophan YT(Xi'an) Biochem Co., Ltd. , https://www.ytherblife.com
Protein Technology Topics: SDS-Polyacrylamide Gel Electrophoresis of Proteins
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