The BioRad DCode Universal Mutation Detection System (BioRad, Hercules, CA, USA) was used for DGGE analyses with 8% (w/v) polyacrylamide gels in 1 × TAE. A 30–50% urea-formamide denaturing gradient (diluted from a 7 M urea and 40% (w/v) formamide stock) yielded optimal fungal sample separation. Gels were run for 17 h at 100 V at 60 °C, after which they were stained with AgNO3 as published previously [15 (link)]. The Quantity One software and a calibrated imaging densitometer GS-710 (Bio-Rad, Hercules, CA, USA) were then used to image and analyze DGGE fingerprint profiles.
Gs 710
Manufactured by Bio-Rad
Sourced in United States
The GS-710 is a benchtop imaging densitometer designed for the quantitative analysis of gels, blots, and other flat samples. It features a high-resolution CCD camera and advanced software for accurate and reproducible image capture and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one software, by Bio-Rad (4 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Dcode universal mutation detection system, by Bio-Rad (1 mentions)
Protein ladder, by Takara Bio (1 mentions)
Eba 1051, by Elpis Biotech (1 mentions)
Image master 2d platinum v5, by GE Healthcare (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Ecl western blotting detection kit, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
Total protein extraction kit, by Boster Bio (1 mentions)
Nf κb p65 primary antibody, by Santa Cruz Biotechnology (1 mentions)
Quantity one 4.6.3 1 d analysis software, by Bio-Rad (1 mentions)
Nitrocellulose filter, by Bio-Rad (1 mentions)
Pierce ecl plus, by Thermo Fisher Scientific (1 mentions)
Caspase 9, by Novus Biologicals (1 mentions)
Phospho erk, by Cell Signaling Technology (1 mentions)
Supersignal west femto chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Phospho bad, by Cell Signaling Technology (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
21 protocols using gs 710
1
Fungal DGGE Profiling using BioRad System
2
SDS-PAGE Analysis of Myosin Samples
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Polyacrylamide gel (12.5%) was used to perform sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Lee et al., 2020 (link)). The extracted myosin samples
(protein concentration, 0.5 mg/mL) were diluted twice (v/v) with sample
buffer (EBA-1051, Elpis Biotech, Daejeon, Korea) and heated at 95°C.
The samples (0.5 mg protein/mL) and protein ladder (3454A, Takara Bio,
Shiga, Japan) were loaded at 10 and 5 μL, respectively. The bands
were stained with a solution containing Coomassie brilliant blue, acetic
acid, and methanol overnight. Then, a buffer containing acetic acid and
methanol was used to de-stain the gels. The gels were scanned at an optical
resolution (63.5 μm/pixel) with a densitometer (GS-710, Bio-Rad
Laboratories) and analyzed using Image Master 2D Platinum v5.0 (GE
Healthcare, formerly Amersham Biosciences, Seoul, Korea).
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Lee et al., 2020 (link)). The extracted myosin samples
(protein concentration, 0.5 mg/mL) were diluted twice (v/v) with sample
buffer (EBA-1051, Elpis Biotech, Daejeon, Korea) and heated at 95°C.
The samples (0.5 mg protein/mL) and protein ladder (3454A, Takara Bio,
Shiga, Japan) were loaded at 10 and 5 μL, respectively. The bands
were stained with a solution containing Coomassie brilliant blue, acetic
acid, and methanol overnight. Then, a buffer containing acetic acid and
methanol was used to de-stain the gels. The gels were scanned at an optical
resolution (63.5 μm/pixel) with a densitometer (GS-710, Bio-Rad
Laboratories) and analyzed using Image Master 2D Platinum v5.0 (GE
Healthcare, formerly Amersham Biosciences, Seoul, Korea).
3
Murine Heart Apoptosis Pathway Analysis
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Murine hearts were processed as previously described (Tocchetti et al., 2014 (link)). Anti-caspase 3, anti-cleaved caspase 3, anti-GAPDH (Cell Signaling Technology), or anti-Actin antibody (Sigma), followed by anti-rabbit, HRP-conjugated IgGs from goat antiserum (Thermo Scientific) were used to detect proteins involved in the apoptotic pathway. The signal from secondary antibodies was visualized by enhanced chemiluminescence detection (ECL western blotting detection kit, Thermo Scientific). The signal intensity of reactive bands was quantitatively measured with a phosphorimager (GS-710, Biorad) or by the open source software ImageJ (NIH, USA).
4
Quantifying NF-κB p65 Protein Levels
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Total proteins were purchased from pancreatic tissue using a total protein extraction kit (Boster, Wuhan, China). Protein concentrations were determined using a commercial kit (Pierce, Rockford, IL, USA). Each 20-μg aliquot of total protein was loaded onto sodium dodecyl sulfate-polyacrylamide gel for electrophoresis and then transferred onto membranes. Following complete protein transfer, the membranes were blocked with 5% milk powder solution for 2 h and incubated with NF-κB p65 primary antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight. After washing the membranes, the secondary antibody (Boster, Wuhan, China) was applied and incubated for 2 h at room temperature (RT). Bands were quantified by a calibrated imaging densitometer (GS-710; Bio-Rad) and analyzed by “Quantity One” software (Bio-Rad). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference.
5
Gel Electrophoresis Protocol for Quantification
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One nanomole of each sample was diluted with 10 μl formamide loading buffer. The samples were heated for 3 min at 95 °C before being loaded onto the gel. The gel was run for 1.5 h at 150 V. For band visualization, the gel was agitated in a 2% solution of methylene blue for 30 min and subsequently destained with water. The gel was scanned with a densitometer (Bio-Rad GS-710, Carlsbad, CA, USA) and analyzed with Quantity One 4.6.3 1-D Analysis Software (Bio-Rad).
6
Western Blot Analysis of TCL1 Signaling
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Equal amounts of protein were electrophoresed in SDS-polyacrylamide gels and blotted onto nitrocellulose filters (Bio-Rad). Membranes were blotted overnight at 4°C with primary antibodies. The employed antibodies were the following: anti-human TCL1 (27D6) from Areta International; Akt (cat. 9272), phospho-Akt (cat. 4058), ERK (cat. 9102), phospho-ERK (cat. 9101), Bcl-2 (cat. 3498), Mcl-1 (cat. 5453), and phospho-Bad (cat. 9295) from Cell Signaling Technology; PARP (cat. NB100-111) from Novus Biologicals; caspase-9 (cat. RB1205P1) by Neo Markers; and IkBα (cat. sc-371), β-actin (cat. sc-130656), anti-mouse IgG-HRP (cat. sc-2005) and anti-rabbit IgG-HRP (cat. sc-2030) from Santa Cruz Biotechnology. For immunodetection, Pierce ECL plus or Super Signal West Femto chemiluminescent substrates (Thermo Scientific) and GE Healthcare films were used. Protein bands were quantified by scanner densitometry (GS-710, Bio-Rad) and analyzed with Quantity One software (Bio-Rad). TCL1 OD values were first normalized on β-actin expression and then on control (ctrl) TCL1/β-actin ratio.
7
Purification and Characterization of Anti-gM Antibody
8
Protein Gel Imaging and Analysis
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The gels were stained with colloidal Coomassie Blue and scanned using a GS710 calibrated imaging densitometer (BioRad, Hemel Hempstead, UK). TIFF format images were analysed using Image Master TM 2D Elite software, version 4.01 (Amersham Pharmacia Biotech, Buckinghamshire, UK).
9
SDS-PAGE Analysis of Protein Samples
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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was
performed using a 12.5% polyacrylamide gel containing 30% acrylamide solution,
1.5 M Tris-HCl (pH 8.8), 0.5 M Tris-HCl (pH 6.8), 10% ammonium persulfate, and
N,N,N’,N’-tetramethyl-ethylenediamine. The digesta sample was
mixed with the same volume of 2° sample buffer composed of 125 mM
Tris-HCl (pH 6.8), 20% glycerol, 2% SDS, 2% mercaptoethanol, and 0.02%
bromophenol blue, and heated at 95°C on a heating block for 90 sec. The
10 μL (74.7 μg protein) of a sample and the 5 μL of protein
molecular markers (9-200 kDa) were loaded. Electrophoretic separation was
performed with the pageRun system (AE-6531 mPAGE, ATTO Co., Tokyo, Japan) by
applying 40 mA for 40 min. The running buffer was composed of 25 mM Tris, 0.1%
SDS, and 192 mM glycine. Proteins in the gels were stained with Coomassie
Brilliant Blue and then destained in a 10% acetic acid solution. The stained gel
was scanned using a GS-710 (Bio-Rad Laboratories Inc, Hercules, CA, USA)
densitometer at an optical resolution of 63.5 μm/pixel.
performed using a 12.5% polyacrylamide gel containing 30% acrylamide solution,
1.5 M Tris-HCl (pH 8.8), 0.5 M Tris-HCl (pH 6.8), 10% ammonium persulfate, and
N,N,N’,N’-tetramethyl-ethylenediamine. The digesta sample was
mixed with the same volume of 2° sample buffer composed of 125 mM
Tris-HCl (pH 6.8), 20% glycerol, 2% SDS, 2% mercaptoethanol, and 0.02%
bromophenol blue, and heated at 95°C on a heating block for 90 sec. The
10 μL (74.7 μg protein) of a sample and the 5 μL of protein
molecular markers (9-200 kDa) were loaded. Electrophoretic separation was
performed with the pageRun system (AE-6531 mPAGE, ATTO Co., Tokyo, Japan) by
applying 40 mA for 40 min. The running buffer was composed of 25 mM Tris, 0.1%
SDS, and 192 mM glycine. Proteins in the gels were stained with Coomassie
Brilliant Blue and then destained in a 10% acetic acid solution. The stained gel
was scanned using a GS-710 (Bio-Rad Laboratories Inc, Hercules, CA, USA)
densitometer at an optical resolution of 63.5 μm/pixel.
10
Hepatic Protein Extraction and Analysis
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Hepatic whole cell lysate protein was extracted utilizing previously described methods [23 (link)]. Protein concentrations of the sample were assessed using the Bradford assay (Bio-Rad, Hercules CA). The protein expression bands were quantified using a densitometer (GS-710 calibrated imaging densitometer, Bio-Rad). The antibodies against acetyl-CoA carboxylase (ACC), stearoyl-CoA desaturase 1 (SCD-1), total- and phosphor- AKT (p-AKT, t-AKT), glucose regulated protein 78 (GRP78/Bip), total- and phosphor- (Thr980) PERK (t-PERK, p-PERK), total- and phosphor- (Ser51) eukaryotic translation initiation factor 2a (t-eIF2a, p-eIF2a), total- and phosphor- (Thr183/Tyr185) JNK (p-JNK, t-JNK), total- and phosphor- ERK (p-ERK, t-ERK) were all purchased from Cell Signaling (Danvers, MA). The antibodies against sterol regulatory element-binding protein-1c (SREBP-1c) and C/EBP homologues protein (CHOP) were purchased from Santa Cruz Biotechnology (Dallas, TX).
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