Experimental procedure
Two-dimensional protein electrophoresis
1) First-direction solid phase pH gradient isoelectric focusing electrophoresis
a. Loading: The first isoelectric focusing is performed by means of in-gel loading. Accurately absorb a certain amount of protein sample (140 ug for silver dyed samples, 1.3 mg for dyed samples), add heavy foaming solution (8 mol/L urea, 2% CHAPS, traces of pentaphenol blue, 20 mmol/L DTT, 0.5% IPG buffer) to a final volume of 350 ul. After mixing, it is evenly added between the positive and negative electrodes of the IPG strip holder. Remove the protective film from the prefabricated IPG strip (pH 3 -10 NL) with the glue facing down and over the sample in the holder; slowly press down on the strip and move back and forth to avoid bubble formation. Cover the oil evenly with 1 ml over the strip and cover.
b. Isoelectric focusing: The strip holder containing the sample and IPG strip is placed on an electrophoresis instrument (IPGphorTM Isoelectric Focusing System) for isoelectric focusing. Isoelectric focusing parameters: the bubble rises 3h at 0V, 10h at 30V, 0.5h at 500V, 0.5h at 2000V, 0.5h at 5000V, and 20h at 8000V .
c. Balance of the strip: After the first-direction electrophoresis is completed, the IPG strip is taken out twice for 15 min each time. Equilibration buffer (50 mol/L Tris buffer pH 8.8, 6 mol/L urea, 30 glycerol, 0.8 g phenol blue, 20 mmol/L DTT, 2% (w/v) SDS) reduces electrical infiltration, Conducive to the transfer of protein from the first direction to the second direction. In the second step of equilibrium, 100 mmol/L iodoacetamide was used in place of DTT to remove the DTT at the first step equilibrium. The balanced IPG strip is used to remove excess balance solution along the edges with filter paper.
2) Second-direction vertical SDS-PAGE
According to the volume of the IPG conversion Kit, a uniform glue with a size of 200 mm×200 mm×1 mm and a concentration of 12.5% ​​T and 3.3% C was prepared. The well-balanced IPG strips were transferred over the gel and blocked with 0.5% agarose running buffer. The electrophoresis buffer system was a Tris-Glycine-SDS system (500 ml, SX, 7.5 g Tris, 2.5 g SDS, 36 g Glycine). Always contact the rubber strip and the two-way adhesive to avoid air bubbles; the sealing temperature should not be too high, and do not introduce air bubbles when sealing.
Electrophoresis parameter setting: 14-15 ° C constant temperature, in terms of each strip
20 mA constant current electrophoresis for 40 min
30 mA constant current electrophoresis to the bottom of the phenol blue electrophoresis.
2. Dyeing
1) Silver staining method: transfer SDS-PAGE after completion of the two-dimensional electrophoresis to fixative solution (40% absolute ethanol, 10% glacial acetic acid), fix for 30 min; discard the fixative for sensitizing solution (30% anhydrous) Ethanol, 6.8% sodium acetate, 0.2% sodium thiosulfate, 0.5% glutaraldehyde) sensitized for 30 min; discard the sensitizing solution, wash twice with 200 ml of double distilled water for 30 min each time; Liquid ((0.25% silver nitrate, 40% formaldehyde) for 20 min, avoiding light during dyeing; discard silver staining solution, wash twice with double distilled water 200 ml for 1 min each time; color solution (2.5% sodium carbonate, 20% formaldehyde) color development 2-5 min, until the protein spots are completely revealed; discard the color developer, immediately change the stop solution (1.46 % EDTA), terminate the color development for 10 min; wash with ultrapure water 3 times, each time 5 min .
2) Coomassie blue staining (test dyeing) method: Transfer the SDS-PAGE after completion of the two-dimensional electrophoresis to a fixing solution (50% absolute ethanol, 10% glacial acetic acid in water), fix for more than 30 min; discard the fixative Adding staining solution (10% glacial acetic acid, G-350 filtered), heat dyeing for 10 min; then transfer to decoloring solution (10% aqueous glacial acetic acid) overnight until the background color is exhausted; ultrapure water is washed.
3. Image analysis
The gel was scanned at 256 and 300 dpi with an ImageScanner optical scanner. Image analysis was performed using Image Master 2D Elite 3.01 analysis software.
4. Peptide mass fingerprinting (PMT) analysis sample preparation
1) Decolorization: Select the target protein spots on the 2D gel, cut the protein spots along the stained edge with a clean anatomical blade, and cut into 1-2 mm film, and cut a piece of protein-free 2D gel as a blank control. After that, shake with a solution containing 50% acetonitrile, 25 mmol/I, NH4CO3 for 20 min, discard the solution, and repeat 1-2 times until the blue color in the film fades.
2) Enzyme digestion: The film was vacuum-dried and dried for 20 min to reduce the volume of complete dehydration. Trypsin was mixed with 25 mmol/L NH4CO3 solution to form 0.01 ug/ul, 10 ul was added to each sample tube, and placed in a refrigerator at 4 °C. Min, the enzyme solution was completely absorbed, and the reaction was sealed at 37 ° C for 12 h.
3) Peptide extraction: add 5% TFA (trifluoroacetic acid) solution 120 ul at 40 ° C for 1 h, aspirate the supernatant, add 2.5% TFA, 50% ACN (acetonitrile) solution 120 ul at 30 ° C for 1 h, aspirate The supernatant was combined and the supernatant was combined and lyophilized.
5. Peptide mass fingerprinting analysis of differential proteins
After lyophilization, 10 ul of 0.5% TFA solution or desalted extract was used, and Bruker's mass spectrometer REFLEX III matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- TOF-MS) for analysis. 1 ul of the target was taken, and another 1 ul of a-cyano-4-trans cinnamic acid dissolved in 0.1% TFA/50% ACN was used as a substrate, and dried at room temperature. The mass spectrometer uses a laser wavelength of 337 nm for reflection detection.
6. Database search preliminary identification of protein
The database search program looks for proteins that match these parameters in the database based on the input isoelectric point of the protein, the molecular weight range, and the skin fingerprint data and other parameters. The search database program is: http://
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