Protein expression was analyzed by SDS-PAGE using 7% gels. Proteins were visualized by Colloidal Coomassie Brilliant Blue staining or Western Blotting. Western Blotting was performed using a tank blot system (BioRad) and proteins were detected with a primary anti-His antibody (Qiagen), anti-PDR5 antibody (Davids Biotechnologie, Regensburg, Germany) or a C219-antibody (Abcam) in combination with a secondary anti-mouse antibody or anti-rabbit antibody, respectively, labeled with horseradish peroxidase (Jackson Immuno Research). Protocols followed the manufacturer’s instructions.
Tank blot system
Manufactured by Bio-Rad
Sourced in Germany, United States, United Kingdom
The Tank blot system is a laboratory equipment used for transferring proteins from polyacrylamide gels to membranes for further analysis. It provides a uniform and efficient transfer of proteins from the gel to the membrane, allowing for the detection and analysis of specific proteins.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti his antibody, by Qiagen (1 mentions)
Horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
Nitrocellulose membrane, by LI COR (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Revert total protein stain, by LI COR (1 mentions)
Odyssey blocking buffer in tbs, by LI COR (1 mentions)
Odyssey fc imager, by LI COR (1 mentions)
Irdye 800cw goat anti rabbit igg, by LI COR (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Phos tag acrylamide, by Fujifilm (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Carl Roth (1 mentions)
Irdye 800cw, by LI COR (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Odyssey classic imaging system, by LI COR (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Irdye 680rd, by LI COR (1 mentions)
5 protocols using tank blot system
1
Protein Expression Analysis by SDS-PAGE and Western Blot
2
Western Blot Analysis of UCP1 in Mouse Adipose Tissues
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Western blot analysis was performed as previously described [25 (link)]. Briefly, proteins were isolated from BAT, SAT and VAT of female and male Hh mice using RIPA lysis buffer and quantified using a BCA assay (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer’s instructions. A total of 5–40 µg of protein was separated using 12% SDS–PAGE (Bio–Rad Laboratories GmbH, Feldkirchen, Germany). The proteins were immunoblotted to a nitrocellulose membrane (LI-COR Biosciences, Lincoln, RI, USA) using a tank blot system (Bio–Rad Laboratories GmbH, Feldkirchen, Germany). The membranes were blocked in Odyssey® blocking buffer in TBS (#927-60001, LI-COR Biosciences) and incubated with the primary antibody rabbit-anti-UCP1 (1:1000, #ab10983 Abcam,) overnight. The secondary antibody IRDye® 800 CW goat anti-rabbit IgG (1:10,000 LI-COR Biosciences, #926-32211) and the Odyssey® Fc imager (LI-COR Biosciences) were used for the detection and quantification of the proteins. Total protein concentrations were determined using REVERT-Total Protein Stain (LI-COR Biosciences), which was used for normalization.
3
Cdk7 Phosphoisoforms Analysis by PhosTag
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For analysis of Cdk7 phosphoisoforms from cell lysates, lysates were resolved on 10% SDS-PAGE gels containing 40 μM PhosTag-Acrylamide (Fujifilm Wako Chemicals) and 80 μM MnCl2. Samples were transferred to nitrocellulose using transfer buffer (25 mM Tris-HCl, 192 mM glycine, pH 8.3, 20% (v/v) methanol, 0.1% SDS) in a tank-blot system (Biorad) at a constant current of 400 mA for 2 h at 4°C. After blotting, immunoblotting was performed as described above.
4
Protein Lysate Preparation and Immunoblotting
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For preparation of protein lysates, harvested cells were incubated on ice for 30 min with the following lysis buffer: 50 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.1% Triton X-100 and 5 mM EDTA containing protease/phosphatase inhibitors (1 mM leupeptin, 1 mM aprotinin and 250 μg/mL sodium vanadate). The BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA) was used to determine the protein concentration of cell lysates. Subsequently, after denaturation in Laemmli SDS sample buffer at 95 °C for 5 min, 30 to 40 µg of each sample were separated on SDS polyacrylamide gels. The tank blot system (Bio-Rad, Hempstead, UK) was used for immunoblotting of separated proteins onto a nitrocellulose membrane (GE Healthcare Life Science, Buckinghamshire, UK). After blotting, membranes were blocked in 5% FCS or skim milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) and 1% BSA for 1 h at room temperature. Used antibodies as well as dilution and incubation conditions are described in the Supplemental File S1 .
5
Western Blot Analysis of Transfected Cells
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HepG2 cells were lysed 22 h after transfection by addition of buffer A (82.25 mM Tris-HCl, 32.9% (w/v) glycerol, 2.6% SDS, pH 6.8). The lysate was mixed 1:4 with buffer A with additional 5% β-mercaptoethanol and was heated to 95 °C for 10 min. Proteins were separated on a 12% SDS-PAGE and blotted on a Nitrocellulose membrane (Carl Roth, #HP40.1) using a tank blot system (Bio-Rad, #1703930). The membrane was blocked with blocking buffer, a 1:1 mixture of TBS-T (50 mM Tris, 150 mM NaCl, 0.05% Tween 20 (w/v), pH 7.4) and Odyssey Blocking Buffer (Li-Cor, #927-40000). Membranes were incubated with primary antibodies in blocking buffer and incubated over night at 4 °C. Primary antibodies used were rabbit polyclonal anti-GFP (Thermo Fisher Scientific, #A-11122) diluted 1:2,000 and mouse monoclonal anti-beta-Actin (Cell Signaling, #3700) diluted 1:5,000. The secondary antibodies donkey anti-mouse coupled to IRDye 680RD (Li-Cor, #926-68072) and donkey anti-rabbit coupled to IRDye 800CW (Li-Cor, #926-32213) were diluted 1:10,000 in blocking buffer. The membrane was imaged using an Odyssey Classic Imaging System (Li-Cor).
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