Glucoamylase activity was assayed using the DNS method. Protein fermentation supernatant samples were mixed with an equal volume of 2× protein loading buffer and boiled for 10 min. The samples were subjected to SDS-PAGE using 4 % stacking gels and 12 % resolving gels in a mini-vertical electrophoresis system (Bio-Rad Laboratories, USA). The gels were stained with CBB-R250.
Mini vertical electrophoresis system
Manufactured by Bio-Rad
Sourced in United States
The Mini-vertical electrophoresis system is a compact and versatile laboratory equipment designed for performing gel electrophoresis. It is used to separate and analyze biomolecules, such as proteins or nucleic acids, based on their size and charge. The system provides a reliable and consistent platform for efficient electrophoresis experiments.
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Lab products found in correlation
Coomassie brilliant blue g 250, by Sangon (1 mentions)
Trypsin, by Promega (1 mentions)
Centrifugal concentrator, by TOMY (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Trypsin, by Merck Group (1 mentions)
Quantity one 4, by Bio-Rad (1 mentions)
Precision plus protein dual color standard, by Bio-Rad (1 mentions)
Mini protean, by Bio-Rad (1 mentions)
Geldoc it, by Analytik Jena (1 mentions)
Protoblue safe, by National Diagnostics (1 mentions)
Bis tris polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
Precision plus protein standard, by Bio-Rad (1 mentions)
6 protocols using mini vertical electrophoresis system
1
Glucoamylase Activity Assay Protocol
2
SDS-PAGE Protein Separation and Digestion
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Before being subjected to SDS–PAGE, the extracted total protein was dissolved in SDS–PAGE loading buffer, boiled for 5 min, centrifuged at 20,000 g for 10 min, loaded on a 5% stacking gel and 12% separating gel, and was run at 15 mA for 30 min and then 30 mA for 2 hr in a mini-vertical electrophoresis system (Bio-Rad, USA). After electrophoresis, the gel was stained overnight in a solution of 0.1% (w/v) Coomassie Brilliant Blue G-250 (Sangon Biotech, Shanghai, China), 30% (v/v) methanol, and 10% (v/v) glacial acetic acid. After decolorization, the gel was analyzed for bands along with a molecular weight marker.
Thereafter, the gel containing all bands was cut into 1 mm3 particles for in-gel digestion: gel particles were washed three times in deionized water and subsequently dehydrated with 100% acetonitrile (ACN) for 10 min. The particles were incubated with 100 mM DTT for 30 min at 56℃. The resulting free thiol (–SH) groups were subsequently alkylated by incubating the samples with 200 mM iodoacetamide for 20 min in the dark. Gels were washed with 25 mM ammonium bicarbonate and dehydrated with 100% ACN sequentially. Thereafter, 10 ng/μl trypsin (Promega, USA) was added and incubated for 20 hr at 37℃ for protein digestion. Supernatants were transferred to fresh tubes for mass spectrometric analysis.
Thereafter, the gel containing all bands was cut into 1 mm3 particles for in-gel digestion: gel particles were washed three times in deionized water and subsequently dehydrated with 100% acetonitrile (ACN) for 10 min. The particles were incubated with 100 mM DTT for 30 min at 56℃. The resulting free thiol (–SH) groups were subsequently alkylated by incubating the samples with 200 mM iodoacetamide for 20 min in the dark. Gels were washed with 25 mM ammonium bicarbonate and dehydrated with 100% ACN sequentially. Thereafter, 10 ng/μl trypsin (Promega, USA) was added and incubated for 20 hr at 37℃ for protein digestion. Supernatants were transferred to fresh tubes for mass spectrometric analysis.
3
Proteome Identification and Characterization
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Ten micrograms of SABH was mixed with 2× protein loading buffer, containing 2× Laemmli sample buffer (Bio-Rad Laboratories, USA) and 2-mercaptoethanol, and denatured by heating at 65 °C for 10 min. The sample was subjected to one-dimensional SDS-PAGE, using 5% stacking and 12% resolving gels, and a mini-vertical electrophoresis system (Bio-Rad Laboratories, USA). The gels were stained with Coomassie brilliant blue G250. An individual lane of the SDS-PAGE gel was segmentally cut along its length into small pieces. To perform in-gel digestion, gel pieces were destained until colourless, using acetonitrile (50%) in 50 mM NH4HCO3. After the destaining solution had been removed, 10 mM dithiothreitol was added to the gel pieces, followed by incubation at 60 °C for 15 min. Proteins were alkylated using 55 mM iodoacetamide in 50 mM NH4HCO3 at room temperature for 30 min in the dark. Following the removal of all solution, the gel pieces were dehydrated by 100% ACN (Sigma-Aldrich, USA) and dried at room temperature. Protein digestion was performed overnight at 37 °C, using 0.1 mg/mL trypsin (Sigma-Aldrich, USA) in 50 mM ammonium bicarbonate. Peptides were extracted in 50% ACN. The resulting supernatant was transferred to microcentrifuge tubes and dried in a centrifugal concentrator (TOMY, Japan) at 45 °C.
4
SDS-PAGE Analysis of Tigriopus flavidus Proteins
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SDS-PAGE was conducted as described in our earlier study [2 (link)]. Briefly, 300 µL of 2 µg/µL T. flavidus extract was mixed with 100 µL of loading buffer, incubated in boiling water for 3 min, cooled at room temperature, and then centrifuged at 8500× g for 5 min. Supernatant (5 µL/lane) was characterized by a polyacrylamide gel (4% stacking and 8% running) on a mini vertical electrophoresis system (Bio-Rad Laboratories, US). After electrophoresis, the gel was incubated in fixing solution (methanol:acetic acid:water, 50:10:40) for 30 min, stained by 0.1% Coomassie brilliant blue for 30 min, and then rinsed by 30% methanol containing 10% acetic acid for 30 min. Contents of the collagen subunits were estimated by Quantity One 4.6.0 (Bio-Rad Laboratories, US).
5
Protein Profiling by SDS-PAGE
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To study protein profiles, SDS-PAGE was performed in a mini vertical electrophoresis system (Bio-Rad, Mini-PROTEAN, CA, USA). Equal quantities of protein (25 µg) from each sample were loaded into 12.5% gels, and Precision Plus Protein Dual Color Standards (Bio-Rad) were incorporated into the gel to determine the molecular weight of the bands.
6
SDS-PAGE Analysis of E. scolopes OMVs
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Samples containing 10 μg of total protein from E. scolopes OMVs were loaded onto a precast 12% bis-tris polyacrylamide gel (Invitrogen), together with a Precision Plus Protein standard (Bio-Rad). The proteins were separated for 1 h at 150 V at 4 °C in a Mini-Vertical Electrophoresis System (Bio-Rad), stained overnight in 90% Protoblue Safe (National Diagnostics,) in ethanol, rinsed in deionized water, and imaged with GelDoc-It (UVP) system.
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