Determination of protein molecular weight (SDS-polyacrylamide gel electrophoresis)

related topics .
, principle
the separation and identification of
proteins
(protein) by polyacrylamide
gel electrophoresis
method, mainly depends on the charge effect and molecular sieve effect. The components of each zone can be determined by comparing with the standard sample. To determine the protein molecular weight of a sample by gel electrophoresis, the charge effect must be removed, so that the migration rate of the protein molecule of the sample depends entirely on the molecular weight. For example, a certain concentration of sodium sulphate (Sodiumdo decylsulfate for short SDS) is added to the electrophoresis system. This anion
surfactant
concentration greater than 1mmol/L to 1. 4gSDS binds to 1g protein molecules to form a complex that makes the protein negatively charged, which far exceeds the original charge differences of protein molecules, thereby reducing or eliminating the natural charge differences of various proteins.ethanol is a reducing agent of the two sulfur bonds in protein molecules, so that
peptides
components are divided into a single sub-unit. SDS breaks the hydrogen bond of a protein. Therefore, when it binds to proteins, it also causes changes in protein composition, and the fluid mechanics and optical properties of this complex indicate that their shape in the aqueous solution is similar to the rod of a long elliptical garden. The short axis of different protein-SDS complexes is the same, about 1. 8nm, and the long axis changes in directly in direct terms with the molecular weight of the protein. Therefore, the migration rate of various SDS-protein complexes in electrophoresis is no longer affected by the original charge and shape, but is separated by the molecular sieve effect of the gel according to the size of the molecule, and its effective migration rate is linearly related to the log of molecular weight. In this way, the molecular weight of the final sample protein can be derived from the standard curve of the standard protein molecular weight of the number of pairs and the migration rate.II, materials,
instruments
and
reagents
:
(i) Materials: wheat buds
(ii) instruments and equipment
: 1. vertical plate electrophoresis
tank and accessories;
(iii) Reagents: 1. Lower glue buffer: 18.17g Tris, 0.4gSDS, soluble in water. Tune pH8.8 with 1mol/L HCl to 100ml. 2. Upper glue buffer: 6.06g Tris, 0.4gSDS, soluble in water, 1mol/L HCl to 6.8, 100ml. 3. Acr/Bis storage fluid: 30g Acr, 0.8g Bis, dissolved to 100ml. 4. 10% ammonium per sulfate, ready to use. 5. Sample buffer: Tris0.6g, glycerin 5ml, SDS1g soluble in water, HCl to pH to 8.0, plus brominated phenol blue 0.1g,base ethanol 2.5ml, fixed to 100ml. 6. Electrode buffer: Tris 3.03g, Gly14.14g, SDS1.0g, soluble in water, with HCL pH to 8.3 tolerance to 1000ml. 7. Preparation of dyeing fluid: 45% methanol, 0.25% Comas bright blue R-250. 8. Decolorizer: 2.5% methanol, 10% acetic acid. 9. 1.5%
agar
: 1.5g agar dissolved in 100ml electrode buffer, heated dissolved. 10. Standard molecular weight protein 3, experimental steps:
1. Installation of electrophoresis tank:
drying
of the two glass plates into the matching plastic jacket, fixed vertically on the electrophoresis tank, surrounded by 1.5% agar seal.
2. Sample treatment: the various standard proteins and samples to be tested are dissolved in the protein treatment solution (concentration 2 mg/ml), heated in boiling water 3 to 4min, to be cooled for sample use.
3. Glue: select the appropriate glue concentration according to the table to make 7.5% of the separation glue, mix it along the long glass plate between the two glass plates (careful not to create bubbles), add to the edge of the short glass plate 3cm, immediately cover 2 to 3mm water layer, static polymerization of about 40min. The mark of good glue polymerization is a clear interface between glue and water. Absorb the moisture of the separation glue, the preparation of the concentrate glue into the separation glue, immediately inserted into the coll glass sample slot template, to concentrate the glue polymerization good backup.
4. Dot: Use a microsyscopic syringe to absorb the standard protein sample fluid 5 μl, in order of molecular weight (small to large) into the concentrated surface in the sample tank, while absorbing the sample fluid to be tested 25 μl, injected into other sample tanks, carefully injected electrode buffer on the sample.
5. Electrophoresis: after the addition of samples, two slots into the electrode buffer, on the power supply (up and down positive), adjust the current of 15mA, when the indicator into the separation glue, the current needs to be increased to 30mA, the voltage constant between 80 to 100 volts, about 3 to 4h, the indicator reached 1 to 2cm from the front, electrophoresis can be terminated.
6. Fix: After the glue is removed, soak 10min in water, dip part of SDS, then soak the glue in a mixture of 25% isopropyl alcohol and 10% acetic acid 1h, the protein is fixed.
7. Staining: Remove the gel plate, determine the length of the adhesive (D1), add the dye dye 2h.
8. Decoloring: put the glue in the decolorizer 0.5h, every 0.5h change the decolorizer, at least 3 times, wait for the blue basically removed, and then immerse in the decolorizer until the protein
stat mass spectrometry
band clear, accurate determination of the length of the glue (D2)., protein standard sample migration rate and sample molecular weight to be tested
based on the relationship between protein molecular weight pair and electrophoresis migration rate, accurate measurement of bromphenol blue and various protein migration distance. The distance of bromphenol blue migration is set at d1, the distance of protein migration is set as d2, and D1 is the length before gel dyeing, and D2 is the length after gel dyeing. According to the following formula to calculate the migration rate of various proteins Rm: Rm- d2d1×D1D2 with the standard protein migration rate of the cross-station, with its corresponding molecular weight pair as the vertical base, draw a standard curve, you can get a straight line, and then according to the migration rate of unknown samples, in the half-to-several scale to find out its corresponding molecular weight. To get a reliable result, the experiment needs to be repeated many times..