Liver samples were homogenized in a lysis buffer using a Potter–Elvehjem homogenizer, and total protein and nuclear extracts were prepared as described previously [7 (link)]. To prepare protein extracts form adipose tissue, the samples were homogenized in a lysis buffer using a TissueLyser LT (Qiagen, Düsseldorf, Germany) at 50 Hz for 10 min, centrifuged at 13,000× g for 15 min at 4 °C, and the supernatant was obtained. Protein content in protein extracts was determined by the Bradford assay [14 (link)]. Western blots were performed using four samples per group, each sample pooled from two animals, as described previously [8 (link)]. To confirm the uniformity of protein loading, blots were incubated with anti-β-actin or anti-β-tubulin antibodies (Sigma-Aldrich, St. Louis, MO, USA), or with an anti-vinculin antibody (Santa Cruz Biotech, Dallas, TX, USA), as a control for total protein extracts, and with an anti-TBP antibody (AbCam, Cambridge, UK) for nuclear protein extracts. A list of antibodies used in Western blot analysis is shown in Table S2 .
Anti tbp antibody
Manufactured by Abcam
Sourced in United Kingdom
The Anti-TBP antibody is a laboratory tool used to detect and study the TATA-binding protein (TBP) in various biological samples. TBP is a critical component of the transcription initiation complex, playing a key role in the regulation of gene expression. This antibody can be used in techniques such as Western blotting, immunohistochemistry, and immunoprecipitation to identify and analyze the presence and distribution of TBP in different cell types and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Anti vinculin antibody, by Santa Cruz Biotechnology (3 mentions)
Anti β actin, by Merck Group (2 mentions)
Immobilion western hrp substrate peroxide solution, by Merck Group (2 mentions)
Immobilon polyvinylidene difluoride transfer membranes, by Merck Group (2 mentions)
Anti β tubulin antibody, by Merck Group (2 mentions)
Tissuelyser lt, by Qiagen (1 mentions)
4 protocols using anti tbp antibody
1
Protein Extraction and Quantification from Liver and Adipose Tissue
2
Protein Extraction and Western Blot Analysis
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Tissue samples and western blot analysis were prepared and performed as described previously.[11 ] Western blots were performed using four samples per group, each sample pooled from two animals. A total of 30 µg of protein extracts (10 µg for PNPLA3 determination) was used. Protein concentrations were determined by the Bradford method.[46 ] To confirm the uniformity of protein loading, blots were incubated with anti‐β‐actin, or anti‐β‐tubulin antibodies (Sigma–Aldrich, St. Louis, MO, USA) or anti‐vinculin antibody (Santa Cruz Biotech, Dallas, TX, USA), as a control for total protein extracts, and with anti‐TBP antibody (AbCam, Cambridge, UK), as a control for nuclear protein extracts. A list of antibodies used in western blot analysis is shown in the Supplementary Material (Table S3 , Supporting Information).
3
Western Blot Analysis of Protein Extracts
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Western blots were performed using three samples per group, each sample was pooled from two animals. A total of 20–30 μg of protein extracts were subjected to SDS-polyacrylamide gel electrophoresis. Proteins were then transferred onto Immobilon polyvinylidene difluoride transfer membranes (Millipore, Billerica, MA, USA), and blocked for 1 h at room temperature, with 5% non-fat milk solution in Tris-buffered saline (TBS) containing 0.1% Tween-20. Membranes were then incubated with specific primary antibodies. Detection was performed using the Immobilion Western HRP substrate Peroxide Solution® (Millipore, Billerica, MA, USA). To confirm the uniformity of protein loading, blots were incubated with anti-β-actin or anti-β-tubulin antibody (Sigma–Aldrich, St. Louis, MO, USA) as a control for total protein extracts, and with anti-TBP antibody (AbCam, Cambridge, UK) for nuclear protein extracts.
4
Quantitative Western Blot Analysis of Liver Proteins
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Liver samples were homogenized in lysis buffer using a Potter-Elvehjem homogenizer, and total protein and nuclear extracts were prepared as described previously [16 (link)]. Protein content was determined by the Bradford assay [31 (link)]. Western blots were performed using four samples per group, each sample pooled from two animals. A total of 20–30 µg of protein extracts was subjected to SDS-polyacrylamide gel electrophoresis. Proteins were then transferred onto Immobilon polyvinylidene difluoride transfer membranes (Millipore, Billerica, MA, USA) and blocked for 1 h at room temperature with a 5% non-fat milk solution in Tris-buffered saline (TBS) containing 0.1% Tween-20. Membranes were then incubated with specific primary antibodies (see the list of antibodies used in the Supplementary Material, Table S2 ). Detection was performed using the Immobilion Western HRP substrate Peroxide Solution® (Millipore, Billerica, MA, USA). To confirm the uniformity of protein loading, blots were incubated with antivinculin antibody (Santa Cruz Biotech, Dallas, TX, USA) as a control for total protein extracts, and with anti-TBP antibody (AbCam, Cambridge, UK) as a control for nuclear protein extracts.
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