Vitamins are a type of trace organic substances that humans and animals must obtain from food in order to maintain normal physiological functions. They play an important role in human growth, metabolism and development. Vitamins do not participate in the formation of human cells in the body, nor do they provide energy for the human body. Vitamins are a class of organic compounds necessary for maintaining good health.
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SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is a method often used in protein analysis. It heats the protein sample together with the ionic detergent sodium dodecyl sulfate (SDS) and mercaptoethanol to denature the protein, the disulfide bond between the peptide chain and the peptide chain is reduced, and the peptide chain is opened. . The opened peptide chain is negatively charged by hydrophobic interaction with SDS, and the peptide chain migrates to the positive electrode in the gel under the action of an electric field during electrophoresis. Peptide chains of different sizes are gradually separated during migration due to the different resistances encountered during migration, and their relative mobility is linear with the logarithm of molecular weight.
pGLO is an expression vector in which the gene for green fluorescent protein (GFP) is cloned after the arabinose promoter. E. coli harboring pGLO was cultured under conditions containing the corresponding inducer and ubiquitin to express GFP, which is an extra band on the SDS-PAGE gel than the same E. coli without the inducer. The expressed protein can also be detected by non-denaturing polyacrylamide gel electrophoresis. Under the violet light, it can be seen that the induced sample has a green fluorescent band on the gel. Exogenous proteins expressed can also be detected by GFP antibody by ** blotting (Western-blotting).
[Instruments, Materials and Reagents]
(1) Instrument
1. Vertical plate electrophoresis tank and matching glass plate, comb
2. Electrophoresis
3. Dry constant temperature incubator
4. Microwave oven
(2) Materials
1. Culture of E. coli transformed with pGLO expression plasmid: induced with arabinose and not induced by arabinose
(three) reagent
1.1.5mo1/L Tris-HCl pH 8.8 (SDS added)
2.0.5mo1/L Tris-HCl pH 6.8 (SDS added)
3.10% SDS
4.30% Acr/Bis 29.2g Acr + 0.8gBis, dilute to 100mL with double distilled water, filter for use, store at 4 °C.
5.10%Ap (stored at -20°C)
6.2x sample buffer
0.5mo1/L Tris-HCl pH6.8 2mL
Glycerin 2mL
20% SDS 2mL
0.1% bromophenol blue 0.5mL
2-?-mercaptoethanol l.0mL
Double distilled water 2.5mL
7.5x electrode buffer
Tris 7.5g
G1y 36 g
SDS 2.5g
Dissolve in double distilled water, dilute to 500mL, dilute 5 times when used
8. Staining solution: 0.2 g Coomassie Brilliant Blue R250 + 84 mL 95% ethanol + 20 mL glacial acetic acid, dilute to 200 mL, and filter for use.
9. Decolorizing solution: Alcohol: Glacial acetic acid: Water = 7.5:7.5:85
[Experimental steps]
1. Assemble the glass plate "sandwich" for glue: make 1.0mm thick glue. Select the glass clips on both sides as a 1mm glass plate, place the strips flat on the table top, place the gray U-shaped silicone clips on top, and place the concave glass with two "ears" on it. On the top, the U-shaped silicone clips are flat and compliantly sandwiched between the two glass sheets. Carefully place the glass "sandwich" on the "basket" and clamp it with the plastic "wedge". Note that the U-shaped silicone clip at the bottom remains flat and compliant during the clamping process. Fill the distilled water in the “sandwich†of the glass plate to check for leaks. If the water leaks, it must be reinstalled.
2. Prepare a 12% separation gel (the gel concentration should be selected according to the molecular weight of the protein to be separated). The formula is as follows:
Double distilled water 3.3 mL
1.5mo1/L Tris-HCl (pH 8.8) 2.5 mL
Acr/Big (30%) 4.0 mL
10% SDS 100 μL
TEMED 10 μL
10% AP 100 μL
Total volume 10 mL (just two pieces of glue)
After mixing, add two glass nips, and carefully add 1cm distilled water on the rubber surface (add water to the surface of the rubber surface to seal the water seal, the purpose is to keep the rubber surface flat, and prevent the glue from contacting with air, affecting the polymerization of the glue), room temperature After placing, the glue is naturally agglomerated (in this case, a clear boundary is visible between the rubber surface and the water seal), and the water seal is poured. At this point, you can start to prepare 4% concentrated glue, the formula is as follows:
Double distilled water 1.8 mL
0.5mo1/L Tris-HCl pH 6.8 0.75mL
Acr/Bis (30%) 0.4 mL
10% SDS 30 μL
TEMED 5μL
10%Ap 30μL
The total volume is 3.0 mL (enough to make two plates of concentrated glue)
After mixing, add to the crevice of the "sandwich" glass plate, and then in the 1mm comb ** concentrated glue, be careful not to leave air bubbles between the comb and the concentrated glue. If you find a large bubble, be sure to remove it (you can insert the comb or finger glass, etc.), otherwise it will affect the electrophoresis results. After the gel has solidified, carefully pull out the comb. If it is found that the interval between the sample holes formed after the comb is pulled out is distorted, carefully arrange it with a syringe needle to make it uniform.
3. Preparation of culture SDS-PAGE samples: Two E. coli samples of L-arabinose-induced and L-arabinose-free were simultaneously prepared. 1.5 mL of the culture was added to a 1.5 mL small centrifuge tube, centrifuged at 12000 r/m for 3 minutes, and the supernatant was removed. Add approximately equal volume of distilled water to the pellet and carefully pipette with a pipette tip until fully suspended until no significant small agglomerates are added. Then add an equal volume of 2x sample buffer to a boiling water bath at 100 °C (or dry) Incubate for 5 min in the incubator). At this point, if the sample is taken with a pipette tip, it will be very viscous and snot-like, because there is a large amount of DNA in the sample. The insulin syringe is used to shoot the sample from a very fine needle, and the DNA molecules are broken several times to make the sample no longer sticky. The sample was centrifuged at 14000 r/m for 5 minutes to precipitate insoluble matter, and the supernatant was carefully pipetted. The processing of electrophoresis samples is directly related to the quality of the electrophoresis results. It must be done carefully, otherwise it will not run well.
4. Agar back cover: After making the glue, remove the glass plate “sandwich†from the shelf and remove the silicone clip between the two glass plates. After removing the strip, a gap will be left at the bottom of the gel. The gap should be filled with 2% melted agar. Otherwise, the air in the gap will be completely eliminated after the glass plate is immersed in the electrode buffer. It will obviously affect the conductivity, and if it is serious, the whole electrophoresis will fail. Be careful not to create bubbles when the agar is sealed.
5. Electrophoresis tank assembly: After the back cover, reinstall the glass plate “sandwich†concave glass on the “basket rackâ€. After fixing, place the whole part in the outer casing of the electrophoresis tank and then add the electrode buffer. When adding liquid, the liquid level in the inner and outer tanks should be substantially equal, and the liquid level of the inner tank liquid should be at least 5 mm higher than the concave glass of the glass plate "sandwich" to ensure conductivity and uniform electric field.
6. Loading: Add the samples in the order in advance and the amount of the sample. In the case where the concentration of the sample is unknown, a gradient can be added to the same sample, that is, the amount of loading per lane is halved. In this way, when doing the second electrophoresis, it can be used as a reference and correctly loaded. The protein molecular weight standard can be added to the middle of the sample well, or it can be added to the second side of the edge (a sample on the side of the edge tends to run obliquely).
7. Electrophoresis: Cover the lid of the electrophoresis tank, connect the two electrodes of the electrophoresis tank to the electrophoresis apparatus (note that the electrodes are not connected correctly), and then turn on the power. The voltage was first adjusted to 80V, and when the sample entered the separation gel, the voltage was adjusted to be constant at 150V. When bromophenol blue moved to about 0.5 cm from the bottom, turn off the power and stop electrophoresis. Remove the glass plate from the electrophoresis tank and carefully pry open the two glass plates with the old phone card to expose the rubber surface. Remove the concentrated gel portion. Do not separate the gel into a plastic box for dyeing.
8. Dyeing and Decolorization: The gel is stained in Coomassie Brilliant Blue staining solution for 20 minutes (slow shaking on a shaker), then the staining solution is removed (the staining solution is recovered, it can be repeated dozens of times), washed several times with tap water, and the gel is removed. The dyed residue in the upper and the plastic box was decolorized by decolorization (slow shaking on a shaker), and the decoloring solution was changed once after 1 hour, and the mixture was shaken overnight. The gelatin protein strip after thorough decolorization is clear and the background is transparent and clean. At this time, the gel can be photographed or scanned with a scanner as a record.
Protein Technology Topics: Detection of Exogenous Protein Expression by SDS-Polyacrylamide Gel Electrophoresis
[Experimental principle]