1 mg of isolated liver rat mitochondria were solubilized with 10% lauryl maltoside, shaken for 30 min at 4 °C, and then centrifugated at 17,500 rpm by 75 min 4 °C. The protein concentration was determined by the Bradford method, and 0.1–0.25 mg protein per well was loaded in 4–12% polyacrylamide gradient gels; therefore, the same amount of mitochondrial protein was added for each condition. For clear native electrophoresis was used 0.01% lauryl maltoside and 0.05% sodium deoxycholate were in the cathode buffer. Gels were run for one hour at 15 mV/gel in a Bio-rad electrophoresis chamber55 . In-gel NADH: NBT oxidoreductase activity (Complex I) was determined by incubating the native gels in 10 mM Tris (pH 7.0), 0.5 mg/mL nitrotetrazolium blue chloride (NBT), and 1 mM NADH. In-gel succinate: NBT oxidoreductase activity (Complex II) was defined by incubating the native gels in 10 mM Tris (pH 7.0), 0.5 mg/mL NBT and 1 mM succinate. In-gel cytochrome c oxidase activity (Complex IV) was determined using 20 mg/mL diaminobenzidine (DAB) and 10 mg/mL cytochrome c in 50 mM phosphate buffer pH 7.4. In-gel ATPase activity (Complex V) was measured by incubating the CN-gel in 270 mM glycine and 35 mM Tris buffer pH 8.4 and added 0.2% Pb(NO3)2, 14 mM MgSO4 and 8 mM ATP56 (link).
Electrophoresis chamber
Manufactured by Bio-Rad
Sourced in United States
The Electrophoresis Chamber is a laboratory instrument designed for the separation and analysis of biomolecules, such as proteins or nucleic acids, using the technique of electrophoresis. It provides a controlled environment for the migration of charged particles through a gel medium under the influence of an electric field.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one software, by Bio-Rad (2 mentions)
Nitrocellulose membrane, by Pall Corporation (2 mentions)
Transfer chamber, by Bio-Rad (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Gelred nucleic acid gel stain, by Biotium (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Thermal cycler, by Eppendorf (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
P akt, by Merck Group (1 mentions)
Caspase 9, by Merck Group (1 mentions)
Caspase 3, by Merck Group (1 mentions)
P pi3k, by Merck Group (1 mentions)
High speed refrigerated centrifuge, by Beckman Coulter (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Facs vantage se flow cytometer, by BD (1 mentions)
Trans blot turbo, by Bio-Rad (1 mentions)
Matrigel, by BD (1 mentions)
Co2 incubator, by Thermo Fisher Scientific (1 mentions)
18 protocols using electrophoresis chamber
1
Mitochondrial Enzyme Activity Assay
2
ERIC-PCR Genotyping of Bacterial Isolates
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ERIC-PCR analysis of the 12 isolates was carried out by using primers ERIC-1R (5′-ATGTAAGCTCCTGGGGGGATTCAC-3′) and ERIC-2 (5′-AAGTAAGTGACTGGGGG GTGAGCG-3′) as previously described [21 (link)]. Then, 50 μL PCR-reaction mixtures were prepared with 25 μL of MyTaq Mix (Bioline Reagents, Ltd., London, UK), 0.7 µM of each primer, 5–50 ng of purified DNA, 3 μM of MgCl2, and 19 µL of molecular-biology-grade water. PCR mixtures were subjected to an initial denaturation (95 °C, 1 min), 35 cycles of denaturation–annealing–elongation (95 °C, 15 s; 46 °C, 15 s; and 72 °C, 10 s), and a final elongation (72 °C, 4 min) in a thermal cycler (Eppendorf, Hamburg, Germany). The amplification products were gel-electrophoresed at 90 V for 60 min in an agarose gel (1.5% w/v) (Pronadisa), dyed with GelRed Nucleic Acid Gel Stain (Biotium, Inc.), in an electrophoresis chamber (BioRad Laboratories, Inc.), and subsequently visualized using the ChemiDoc Imaging System (BioRad Laboratories, Inc.), with HyperLadder 100 bp (Bioline Reagents, Ltd.) as molecular weight marker. ERIC-PCR type analysis, clustering, and dendrogram construction were performed by using the Phoretix v.5.0 software (Nonlinear Dynamics Ltd., Newcastle-upon-Tyne, UK).
3
Fecal DNA Extraction Using QIAGEN DNeasy Kit
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The QIAGEN DNeasy Blood and Tissue kit was used to extract the fecal DNA using a UV laminar flow hood with all sterility protocols. The DNA extraction products were run on 1.2% agarose gels at 80 V for 45 min in a Bio-Rad electrophoresis chamber to visualize the presence of DNA. Visualization was carried out on a GelMaxTM photodocumenter (UVP®, Upland, CA, USA). The concentration and quality of DNA obtained from the samples was measured on a Qubit® 3.0. (Invitrogen, Carlsbad, CA, USA).
4
Western Blot Analysis of LdcI Protein
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SDS-PAGE was performed with a Bio-Rad electrophoresis chamber using standard 12% reducing SDS-PAGE gels. Proteins were transferred to a nitrocellulose membrane (Bio-Rad) using a Trans-Blot Turbo Transfer System (Bio-Rad). The membrane was blocked for 1 h using 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) buffer supplemented with 0.1% Tween (TBS-T). Afterward, the membrane was incubated for 1 h with an anti-LdcI antibody (Qalam-Antibodies) in BSA/TBS-T (1:5,000). The membrane was subsequently washed 3 × 10 min in TBS-T and incubated for 1 h with horseradish peroxidase (HRP)-coupled anti-rabbit antibody (1:10,000 in BSA/TBS-T). Finally the membrane was washed 3 × 10 min in TBS-T. The membrane was rinsed once with TBS, prior to detection of antibody-labeled proteins using ECL reagent (GE Heathcare). Incubation and detection of antibody were performed at 25 °C.
5
A549 Cell Culture and Characterization
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A549 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, Manassas). Dulbecco’s Modified Eagle’s medium (DMEM), fetal calf serum, and pancreatin were purchased from Invitrogen (Invitrogen, Waltham, MA, USA). Bicinchoninic acid (BCA) protein quantification kit was purchased from Pierce (PIERCE, Illinois, USA). MatrigelTM basement membrane matrix, Transwell chamber and Matrigel and FACS Vantage SE flow cytometer were purchased from BD Biosciences (BD Biosciences, NJ, USA). Antibodies for Survivin, PCNA, Caspase3, Caspase9, PI3K, p-PI3K, AKT and p-AKT were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). HRP-labeled goat anti-rat secondary antibody was purchased from Santa Cruz (Santa Cruz Biotechnology Dallas, TX, USA). Apoptosis detection kit (Annexin PE/7-AAD) was purchased from Kaiji (Nanjing Kaiji Biological Co., Ltd., China). High speed refrigerated centrifuge was from Beckman (Beckman, Coulter, CA, USA). CO2 incubator was from Thermo (Thermo Fisher Scientific, Waltham, MA, USA). Electrophoresis chamber and Trans-Blot Turbo were purchased from Bio-Rad (Bio-Rad, Hercules, CA, USA). Gel imaging system GDS-800 was purchased from UVP (1UVP, LLC, Upland, CA, USA).
6
Agarose Gel Electrophoresis DNA Visualization
7
Hypoxia Modulates Lactate Metabolism
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HR-2 cells were grown in DMEM-S in 10-cm dishes for 24 h with 0.20 mM oxygen before they were moved to lower oxygen concentrations (0.04, 0.02, and 0.005 mM) for 48 h before harvest. At the time of harvest, cells were scraped into radioimmunoprecipitation lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) and sonicated. Protein levels in the lysates were quantified by BCA (Thermo Fisher Scientific). Western blot analysis was conducted with an electrophoresis chamber and semi-dry turbo transfer system (BioRad, Hercules, CA, USA). The antibodies used were: LDHA: mAB #3582, P-LDHA pAB #8176, HSP90 (mAB #4877) (Cell Signaling Technology, Danvers, MA, USA); MCT-1 (pAB: T-19, sc-14917) and MCT-4 (pAB: H-90, sc-50329) (Santa Cruz Biotechnology, Dallas, TX, USA).
8
Comet Assay for DNA Damage Analysis
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The “Comet Assay” was performed as previously described [27 (link)]. Specifically, zebrafish and cavefish cells were cultured according to former reports. For the irradiation experiments, regularly passaged cells were transferred to 6-well plates at a concentration of 5×105 cells/mL, in a total volume of 2 mL per well. Cells were incubated overnight in the dark according to specific culturing conditions. After 24h, cells were exposed to specific doses of UV radiation (160 and 640 J/m2), using a UV-strata linker (Stratagene). A non-exposed plate served as negative control. Following irradiation, cells were returned to the incubator and sampled at specific time points (0, 1, 2, and 4h) in quadruplicates. Sampled cells were detached by trypsinization and embedded in 0.7% low-melting agarose on microscope slides and lysed in a slide chamber containing alkaline lysis buffer at 4°C overnight. Subsequently, slides were transferred to the electrophoresis chamber (Bio-Rad), filled with ice-cold buffer. Electrophoresis was conducted at 25 V and 0.3 A for 20 min. Finally, slides were neutralized, stained with ethidium bromide (EtBr) and then imaged using an epifluorescence microscope (Zeiss Axiostar) equipped with a green light excitation filter of 518 nm. Images were analyzed according to the Olive Tail Moment system [62 (link)].
9
Protein Separation and Detection Protocol
10
SDS-PAGE Characterization of Protein Fractions
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The protein fractions in the concentrate were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli [19 (link)]. A 10% polyacrylamide gel was used for high molecular weights (36–200 kDa Sigma Marker Sigma) and a 12% gel for low molecular weights (20–66 kDa Sigma Marker). The solution, with 1 and 2 mg/mL PC (pH 12 ± 0.1), was mixed with buffer in a 1 : 1 (v/v) ratio and heated for 3 min in boiling water. Gels were loaded with the treated sample (20 mL) and with molecular weight markers (4 mL) and were run in an electrophoresis chamber (Bio-Rad) at 100 volts for 90–120 min. After this, gels were stained overnight with Coomassie blue solution and washed with a 10 : 10 : 80% (v/v/v) methanol/acetic acid/water solution. The washed gels were scanned in a densitometer (Bio-Rad, Model GS700) and the molecular weights of the separate bands were determined with the Quantity-One software (Bio-Rad).
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