Pulled down proteins were separated on Mini-PROTEAN® TGXTM 4–20% precast gels (BIO-RAD Laboratories, Inc. USA) and then transferred onto a 0.2 µm nitrocellulose membrane by Trans-Blot® TurboTM Transfer Pack (BIO-RAD Laboratories, Inc. USA) and Transfer Blot Turbo Transfer System (BIO-RAD). Each membrane was blocked with 5% BSA in PBS-T. A rabbit-anti-HLTF antibody (Abcam, ab17984) and rabbit-anti-heparanase antibody (InSight Pharmaceuticals, Rehovot, Israel) were applied for the assay. After washing, membrane was incubated with HRP-conjugated anti-rabbit secondary antibody. Detection was performed by applying enhanced chemiluminescence (EZ-ECL, Biological Industries, Israel).
Mini protean tgxtm 4 20 precast gels
Manufactured by Bio-Rad
Sourced in United States
The Mini-PROTEAN® TGXTM 4–20% precast gels are electrophoresis gels designed for protein separation. They feature a 4-20% polyacrylamide gradient, which allows for the separation of a wide range of protein sizes.
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Lab products found in correlation
Ab17984, by Abcam (1 mentions)
Trans blot turbo transfer pack, by Bio-Rad (1 mentions)
Ez ecl, by Sartorius (1 mentions)
Transfer blot turbo transfer system, by Bio-Rad (1 mentions)
Imperial protein stain, by Thermo Fisher Scientific (1 mentions)
Q exactive plus, by Thermo Fisher Scientific (1 mentions)
Mascot, by Matrix Science (1 mentions)
Protean ief cell, by Bio-Rad (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Precision plus protein unstained standard, by Bio-Rad (1 mentions)
4 protocols using mini protean tgxtm 4 20 precast gels
1
Western Blot Analysis of HLTF and Heparanase
2
Protein Identification and Quantification
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Proteins, which were eluted by the DNA pull-down assay, were run into a Mini-PROTEAN® TGXTM 4%-20% precast Gels (BIO-RAD Laboratories, Inc. USA) and stained with ImperialTM Protein Stain (Thermo Scientific, Rockford, IL, USA). The stained gel pieces were digested with trypsin, and then analyzed by LC-MS/MS on Q-Exactive plus (Thermo) and identified by Discoverer software (Discoverer 1.4) with two search algorithms: Sequest (Thermo) and Mascot (Matrix Science) against the human proteome from the Uniprot database, and a decoy database. All the identified peptides were filtered with high confidence, top rank, mass accuracy, and a minimum of two peptides. High-confidence peptides have passed the 1% FDR (false discovery rate) threshold. Semi quantitation was done by calculating the peak area of each peptide. The area of the protein is the average of the three most intense peptides from each protein.
3
Proteomic Analysis of DHEA and S-P Effects
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Whole extract of, intact or treated parasites with DHEA (10 μM), S-P (800 μM) and DHEA / S-P (10 / 800 μM) for 30 minutes, was obtained by lysis in 2D sample buffer;
extracts were centrifuged at 10 000 rpm, soluble fractions were quantified in a Nanodrop TM 2000 (Thermo Scientific) at 280 nm. 100 μg of whole extracts, contained in 125 μL of rehydratation buffer, were load on ImmobilineTM DryStrip pH 3-10, 7 cm strips (GE Healthcare).
After 16 h of passive rehydratation, isoelectric focus was performed in a Protean IEF Cell (Bio-Rad Laboratories, Firmware Version: 1.80) by the supplier specifications.
Strips were equilibrated in an equilibrium buffer (6 M urea, 0.375 M Tris-HCl pH 8.8, 2% SDS, 20% glycerol) with 0.5% dithiothreitol (DTT) for 10 min, and then with equilibrium buffer with 2.5 % iodoacetamide (IAA) for 10 min; strips were loaded in polyacrylamide precast gels (Mini-PROTEAN®TGXTM Precast Gels 4-20%, Bio-Rad)
and electrophoresis was performed at 100 V; then gel were stained with silver nitrate and scanned in a HP Scanjet G4050 scanner.
extracts were centrifuged at 10 000 rpm, soluble fractions were quantified in a Nanodrop TM 2000 (Thermo Scientific) at 280 nm. 100 μg of whole extracts, contained in 125 μL of rehydratation buffer, were load on ImmobilineTM DryStrip pH 3-10, 7 cm strips (GE Healthcare).
After 16 h of passive rehydratation, isoelectric focus was performed in a Protean IEF Cell (Bio-Rad Laboratories, Firmware Version: 1.80) by the supplier specifications.
Strips were equilibrated in an equilibrium buffer (6 M urea, 0.375 M Tris-HCl pH 8.8, 2% SDS, 20% glycerol) with 0.5% dithiothreitol (DTT) for 10 min, and then with equilibrium buffer with 2.5 % iodoacetamide (IAA) for 10 min; strips were loaded in polyacrylamide precast gels (Mini-PROTEAN®TGXTM Precast Gels 4-20%, Bio-Rad)
and electrophoresis was performed at 100 V; then gel were stained with silver nitrate and scanned in a HP Scanjet G4050 scanner.
4
In vitro Ganoderma Protein Digestibility
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In vitro protein digestibility studies of Ganoderma extracts were performed in simulated gastric fluid (SGF) at 37 °C for 60 min according to the procedure previously described by Jimenez et al. in 2013 [22 (link)]. Blank tests without proteins and with BSA were performed to optimize experimental conditions. Thereafter, 18 µL of the crude protein extracts or the samples obtained from in vitro digestion were incubated in SGF at 100 °C for 5 min with 6 µL of 4 × Laemmli sample buffer (Bio-Rad Labs. Alcobendas, Spain). Then, 20 µL of each sample was loaded into Mini-PROTEAN TGXTM Precast Gels 4–20% (Bio-Rad Labs. Alcobendas, Spain). Precision Plus Protein ™ Unstained Standards (Bio-Rad Labs. Alcobendas, Spain) was used as standard containing a mixture of ten Strep-tagged recombinant proteins (10–250 kDa), including three reference bands (25, 50, and 75 kDa). Electrophoresis was carried out at 20 °C and 25 mA per gel, using a buffer containing 25 mM Tris–HCl (pH 8.3), 192 mM glycine, and 0.1% (w/v) SDS. The gels were then stained overnight with a solution containing 1% (w/v) Coomassie Brilliant Blue R-250 [22 (link)].
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