Cell lysates were prepared using lysis buffer (1% SDS, 1 mM Na-vanadate, 10 mM Tris-HCl pH 7.4) in the presence of 0.1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail. Proteins (40 μg) were subjected to SDS-PAGE and electroblotting on nitrocellulose membranes, which were probed with specific primary and HRP-conjugated secondary antibodies. Immunoblots were revealed by enhanced chemiluminescence reagent (Bio-Rad). Images were acquired using the VersaDoc 1000 imaging system and individual band densities were integrated by Quantity One software (BioRad).
Versa doc 1000 imaging system
Manufactured by Bio-Rad
Sourced in United States
The Versa Doc 1000 Imaging System is a versatile device designed for capturing high-quality images of various biological samples. It utilizes advanced imaging technologies to provide clear and detailed visualizations of gels, membranes, and other samples. The system is capable of capturing images in a range of wavelengths, including visible, UV, and chemiluminescent, making it a versatile tool for various applications in the life sciences and research fields.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one software, by Bio-Rad (3 mentions)
Enhanced chemiluminescence reagent, by Bio-Rad (2 mentions)
Quantity one software package, by Bio-Rad (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
β actin, by Abcam (1 mentions)
Chemiluminescence system, by Thermo Fisher Scientific (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Hyperfilm ecl, by GE Healthcare (1 mentions)
Mcp257, by Enzo Life Sciences (1 mentions)
Mcp196, by Enzo Life Sciences (1 mentions)
Mcp79, by Enzo Life Sciences (1 mentions)
Immobilon p, by Merck Group (1 mentions)
6 protocols using versa doc 1000 imaging system
1
Protein Immunoblot Analysis Protocol
2
Electrophoretic Analysis of PCR Products
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We analysed 10 μl out of 25 μl of a PCR mixture by electrophoresis (2 % agarose gels consisting of 0.5 μg/ml of ethidium bromide, 100 V, for 1 h in 1× TAE buffer). We studied and archived gel images using Versa Doc 1000 Imaging System (Bio-Rad Laboratories, Hercules, USA). The patterns we obtained from the electropherograms were converted and analysed using the Quantity One software package, version 4.3.1 (Bio-Rad, San Francisco, CA, USA). Band positions in each gel were normalised using the DNA ladder (the molecular DNA size marker: 100–1000 bp, BLIRT SA, DNA Gdańsk, POLAND). Band matching and isolate similarity were accomplished using the Dice band-based coefficient of similarity. Tolerance and optimisations settings were 2.0 % for both pehX-LM PCR/XbaI and PCR MP. A dendrogram was constructed using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA).
3
Quantification of Bile Acid Receptors in Muscle and Adipose
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Membrane G protein‐coupled bile acid receptors (GPBAR1/TGR5) were extracted from fresh muscle and adipose tissue samples, and measured by Western blotting. Proteins were resolved by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, and transferred onto 0.45‐μm polyvinylidene fluoride membranes. Membranes were blocked with blocking buffer for 2 h and probed with anti‐TGR5 antibody (Abcam, Cambridge, UK) at 1:10,000 dilution and anti‐β‐Actin (Cell Signaling Technology, Danvers, MA, USA) at 1:10,000 dilution overnight at 4°C. After washing with Wash Buffer three times, the membranes were incubated for 1 h at room temperature with goat anti‐rabbit antibody (Zsgb‐bio, Beijing, China) at 1:10,000. The relative concentration of protein was quantified by densitometry using the Versa Doc 1000 Imaging System and Quantity One 4.4 software (Bio‐Rad, Hercules, CA, USA).
4
Protein Extraction and Immunoblotting
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To prepare protein extracts, cells were lysed in RIPA buffer in the presence of a cocktail of protease inhibitors. To this end, the culture medium was removed from the cell culture plate and cells were rinsed with chilled PBS (Phosphate Buffered Saline) 1× buffer. PBS was then removed and cells were pelleted by centrifugation. Washed cellular pellet fractions were suspended in RIPA buffer. After the freezing and thawing procedure, the cells were centrifuged for 5 min at 10.000 rpm. Proteins were then subjected to SDS-PAGE and electroblotting on nitrocellulose membranes, which were probed with specific primary antibodies for citrate carrier (CiC; Abcam, rabbit 1:1000), ATP-citrate lyase (ACLY; Abcam, rabbit 1:1000), acetyl-CoA carboxylase (ACC; Cell Signaling, Rabbit 1:1000), fatty acid synthase (FAS; Cell Signaling, Rabbit 1:1000), and β-actin (Abcam, mouse 1:25000). Immunoblots were revealed by enhanced chemiluminescence reagent (Bio-Rad). Images were acquired using the VersaDoc 1000 imaging system and individual band densities were integrated by Quantity One software (BioRad).
5
Quantifying DARPP-32 Phosphorylation in Rat Brain
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NAcS was identified using the Rat Brain Atlas as corresponding to plates 10–12 [42] and excised using rapid head-freeze technique [38 (link), 43 (link)]. Tissues were solubilized in boiling 1% sodium dodecyl sulfate (SDS) and 50 mM NaF, and protein content was determined by a modified Lowry protein assay method (DC protein assay, Bio-Rad Laboratories, Hercules, CA, USA). Western blot analysis was performed as previously described [44 (link), 45 (link)]. Briefly, proteins (30 μg) were resolved into 10% SDS–PAGE gels and electro-transferred onto nitrocellulose membranes. Membranes were incubated with anti-phospho-Thr34 DARPP-32 (Thr34), anti-DARPP-32 (Cell Signaling Technology, Beverly, MA, USA) and anti-β-actin antibodies. To control for equal loading, membranes incubated with anti-DARPP-32 were stripped and reprobed with anti-β-actin, and membranes incubated with anti-phospho-Thr34 DARPP-32 were stripped and reprobed with anti-DARPP-32 antibody. Detection of proteins was performed using a chemiluminescence system (Pierce Biotechnology Inc., Rockford, IL, USA) and quantified with the Versa Doc 1000 Imaging System (Bio-Rad Laboratories). Total DARPP-32 levels were normalized to those of β-actin, phospho-protein levels were normalized to those of DARPP-32.
6
Immunoblot Analysis of Proteasome Subunits
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Immunoblot analyses of α and β-subunits of constitutive and immuno proteasomes, α-tubulin, free ubiquitin and polyubiquitinated proteins were performed as previously described21 (link),54 (link). Briefly, extracts were resolved by 18% (for free ubiquitin) or 12% (for polyubiquitinated conjugates and all other antigens) SDS-PAGE gel and transferred on Nitocellulose (Sigma-Aldrich, St. Louis, MO, USA, for polyubiquitinated conjugates) or Immobilon®-P (Merck Millipore, Darmstadt, Germany, for all other antigens) transfer membranes. Nitrocellulose membrane was boiled for 5 min to unmask poly-Ub antigens and so make more sensitive and quantitative the immunoblot. The membranes were then incubated in blocking buffer (5% BSA, 0.1%Tween-20 in 1 × PBS), followed by incubation with mAb anti-ubiquitin (P4D1, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), anti α-tubulin (T5168, Sigma-Aldrich, St. Louis, MO, USA), anti-α3, -α4, -α5 (MCP196, MCP79, MCP257, Enzo Life Sciences, Farmingdale, NY, USA), anti-β1, -β2, -β5, -β1i -β2i, -β5i (gift of Prof. S. Ferrone, Harvard Medical School, Boston, MA, USA). Bound antibodies were visualized using the ECL technique and bands were detected with Hyperfilm™ ECL™ (GE Healthcare). Densitometric analysis was performed with a VersaDoc 1000 Imaging System and Quantity One software (Bio-Rad, Hercules, CA, USA).
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