/tools/mwcal/
General method:
Determination of molecular weight of protein by SDS- polyacrylamide gel electrophoresis
[principle]
SDS)- polyacrylamide gel electrophoresis is a new technique developed by Weber and Osborn in Shapiro et al. in the late 1960s. This method for determining the molecular weight of protein has the advantages of rapidity, convenience and simple equipment.
In general electrophoresis methods, the electrophoretic mobility of protein mainly depends on the difference of its net charge, molecular size (i.e. molecular weight) and shape at a certain PH, while SDS- polyacrylamide gel electrophoresis mainly depends on the molecular weight of most protein, and has nothing to do with the original charge and shape.
SDS is an anionic surfactant. Under certain conditions, it can open protein hydrogen bonds and hydrophobic bonds, and combine with these protein molecules in proportion to form negatively charged protein -SDS complexes. Generally, protein binding 1.4g SDS per gram. The combination of SDS and protein makes the protein -SDS complexes have the same negative charge, far exceeding the original charge of protein, thus covering up the original charge difference between protein. In aqueous solution, protein -SDS complex has the same conformation, which is similar to cigar-shaped long elliptical rod (the short axis is 1.8nm, and the long axis is proportional to the molecular weight of protein), thus overcoming the original shape difference between protein. In this way, the mobility of protein -SDS complex in the gel is no longer affected by the original charge and shape, but only a function of protein molecular weight. The relationship between protein molecular weight and electrophoretic mobility can be expressed by the following formula:
lgMr = K–BM
Where Mr is molecular weight; K is a constant; B is the slope; M is liquidity.
Therefore, to determine the molecular weight of protein by this method, we only need to know the molecular weight according to the position of protein to be measured in the LGMR mobility diagram of the standard protein with known molecular weight.
The molecular weight measured by this method, except single-chain protein, is not the complete molecular weight of natural protein, but the molecular weight of the subunits or peptide chains that make up these protein. This method is not suitable for protein with abnormal charge or conformation, or protein with large auxiliary groups, such as histone F 1 and some glycoproteins. It is not suitable for some structural proteins, such as collagen.
SDS- polyacrylamide gel electrophoresis can be divided into SDS- continuous system electrophoresis and SDS- discontinuous system electrophoresis according to the difference of buffer, pH value and gel pore size in gel electrophoresis system. According to the gel shape and electrophoresis mode, it can be divided into SDS- polyacrylamide gel vertical tube electrophoresis and SDS- polyacrylamide gel vertical plate electrophoresis.
In this experiment, SDS- discontinuous vertical plate operation method was used. Through experiments, students can master the principles and techniques of SDS- polyacrylamide gel vertical plate electrophoresis, including gel preparation, gel filling, sample addition, gel stripping and fixed dyeing, and learn to determine the molecular weight of protein by these methods.
[Methods and Steps]
1. Preparation of Sample Adding Solution
1, preparation of standard protein sample solution Weigh 0.5 ~ 1 mg of cytochrome, chymotrypsinogen, pepsin, ovalbumin and bovine serum albumin, put them in a small centrifuge tube (Eppendorf tube), add sample solution 1mL, fully dissolve them, and centrifuge to remove any insoluble matter. Heat treatment in boiling water bath for 3min, and cooling for later use.
2. Preparation of protein sample solution to be tested.
According to the existence of the protein sample to be tested, the protein sample was quantitatively analyzed.
(1) solid
For the protein sample to be tested, if it is a salt-free pure protein solid preparation, the preparation method of the sample adding solution is the same as that of the standard protein sample adding solution; If it is a solid preparation of pure protein containing salt, it should be fully dissolved with water, dialyzed against dialysis buffer 12 ~ 16h, and then 0.5mL (protein concentration should be 0.5 ~ 1 mg/ml) should be sucked, placed in an Eppendorf tube, added with the same volume of concentrated sample solution, heat-treated in boiling water bath for 3 minutes, and cooled for later use.
(2) liquid
If the protein sample to be detected is a salt-free pure protein liquid preparation, the same volume of concentrated sample solution is added, and then heat treatment is carried out; If it is a liquid preparation containing salt, dialysis is needed first.
Second, the preparation of gel
1, preparation of gel plate
(1) washing plate
After diluting the detergent with warm water, soak it in sponge to scrub the glass plate, then rinse it with tap water and dry it with alcohol.
(2) Install the rubber plate
Before gluing, align the glass plate with the edges of plastic fillet and concave ceramic plate, seal it with sealant tape, clamp both ends with plastic clips or iron clips, and ensure sealing, and put it on the fixing frame.
2. Preparation of separation glue and concentrated glue solution
ingredient
Separation gel/ml
Concentrated gel/ml
Gel stock solution
2.5
0.26
Separate gel buffer (pH8.8)
1.9
—
Concentrated gel buffer (pH6.8)
—
0.5
10% sodium dodecyl sulfate
0.075
0.02
Temed
0.026
0.02
Double distilled water
3.05
1.22
10% ammonium persulfate
0.0 13
0.02
total volume
7.6
2
Note: ① Finally, add 10% ammonium persulfate solution, mix to make gel solution, and pour the gel quickly.
(2) Acrylamide and methylene bisacrylamide are nerve agents, which are irritating to the skin. So be careful not to inhale and touch the skin. Fully polymerized polyacrylamide gel is non-toxic.
3, preparing isolation glue
Place the rubber board vertically, insert the sample comb with corresponding thickness, mark the lower edge line of the comb at 1cm, and remove the sample comb. Slowly add the prepared isolation glue solution between the glue making plates until the liquid level reaches the mark. Close to the adhesive interface with a dropper, carefully and slowly cover the n-butanol layer with a thickness of about 0.5cm to prevent the solution from evaporating and ensure the smooth surface of the adhesive.
4, making concentrated glue
After polymerization, pour off n-butanol, rinse the rubber surface twice with distilled water, and suck off the residual liquid with filter paper. Add the concentrated glue solution to the separating glue surface with a dropper, fill the glue board and insert the sample comb.
(3) electrophoresis
1. Install the electrophoresis tank
After the concentrated adhesive is polymerized, remove the comb, sealing tape and six iron clips. The rub plate, that inner core of the electrophoresis tank and another pair of rub plates are sequentially put into the tank. The concave ceramic plate of the dual-system rubber plate should be in contact with the inner core of the electrophoresis tank. Then insert wedge-shaped plates to fix two sets of rubber-making plates. If only one rubber plate is used for each electrophoresis, the other set of rubber plates must be replaced by the plexiglass plate provided.
Step 2 add samples
Fill the inner and outer tanks with buffer solution so that the water level of the inner and outer tanks exceeds the gap between concave plates but is lower than the upper edge of plastic plates. Add the sample into the comb hole with a microsampler.
3. Electrophoresis
Cover the upper cover, and electrophorese at 80 V ~ 100 V for about 3 hours until the electrophoresis front indicated by bromophenol blue reaches the lower edge of the rubber plate, and turn off the power supply. Then pull out the wedge plate and take off the rubber wrench.
(4) dyeing and decoloring
Gently pry the white plastic plate away from the ceramic plate with a blade or thin plate, cut off the separation glue along the junction of the separation glue and the concentrated glue with a blade, and cut a small corner in the upper left corner of the separation glue to mark the sample order. Then put on rubber gloves and carefully move the release glue into the dyeing container. Add 100mL coomassie brilliant blue staining solution (containing 0.25% coomassie brilliant blue R-250, 30% ethanol and 10% acetic acid aqueous solution) into the staining container, cover it, and dye it on a shaker for 2 hours.
Pour the dye solution back into the storage bottle (reusable). Add 100mL decolorizing solution (10% acetic acid, 30% ethanol) to the staining dish, and shake well to decolorize until the protein band is clear.
[Results and Calculation]
1. Gel Stripped off the background color, the color band is clearly visible. According to the real sample, trace or shoot the electrophoresis map.
2. According to the ratio of the migration distance of each protein to the dye migration distance, the relative mobility mr of each protein is calculated, and then the LGmr ~ mr diagram is drawn, from which the molecular weight corresponding to the unknown protein Mr is calculated.