Dopamine release was measured with a Dopamine Research ELISA (ImmuSmol, Bordeaux, France). Brain tissue extracts were collected from the nucleus accumbens of animals that were fed the control chow diet (n = 8) or the quercetin-rich chow diet (n = 8) by using an extraction buffer [0.01 N HCl, 1 mM EDTA, 4 mM sodium metabisulfite, pH (7.0)] with Roche cOmplete Mini tablets (Roche, Mannheim, Germany) according to the manufacturer's protocol. BDNF levels were measured with a Mature BDNF Rapid ELISA Kit (Bisensis Pty Ltd., Thebarton, Australia). Additionally, brain tissue extracts were collected from the nucleus accumbens of animals that were fed the control chow diet (n = 8) or the quercetin-rich chow diet (n = 8) by using an acid extraction buffer [50 mM sodium acetate, 1 M NaCl, 0.1% Triton X-100, pH (4.0)] with Roche cOmplete Mini tablets (Roche, Mannheim, Germany) according to the manufacturer's protocol.
Complete mini tablet
Manufactured by Roche
Sourced in Germany, United States, Switzerland
The Complete Mini Tablets is a laboratory equipment product designed for precise and efficient tablet production. It serves as a compact and versatile solution for small-scale tablet manufacturing operations. The core function of this product is to facilitate the creation of tablets in a controlled and consistent manner, enabling researchers and scientists to conduct their work efficiently.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Immobilon p, by Merck Group (3 mentions)
Phosstop, by Roche (3 mentions)
Pvdf membrane, by Thermo Fisher Scientific (3 mentions)
Image lab software, by Bio-Rad (3 mentions)
Bca protein assay, by Thermo Fisher Scientific (3 mentions)
Ab8245, by Abcam (3 mentions)
Anti trkb, by Abcam (2 mentions)
Anti bdnf, by Abcam (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Las 3000, by Fujifilm (2 mentions)
Ripa buffer, by Roche (2 mentions)
Rtma 2, by Eppendorf (2 mentions)
Bmab 20, by Eppendorf (2 mentions)
Bioruptor pico, by Diagenode (2 mentions)
Complete mini protease inhibitor tablet, by Roche (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Epr1292, by BD (2 mentions)
Ep801y, by Cell Signaling Technology (2 mentions)
71 protocols using complete mini tablet
1
Quercetin's Impact on Dopamine and BDNF
2
CSF Protein Extraction and Trypsin Digestion
3
Renal Antioxidant Enzymes Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Renal cortex (100 mg) was homogenized in Eppendorf tubes with 1 mL of cold phosphate buffer (50 mM, pH 7) and a protease inhibitor mixture (Complete Mini Tablets, Roche). One tablet per each 10 mL of buffer solution [reference number SKU 11697498001, Sigma–Aldrich Chem. Co., St. Louis, MO, USA] was added at 4 °C and 10,000 r.p.m., in three cycles. Afterwards, 1 mL of each homogenate were centrifuged at 10,500 r.p.m. and 4 °C/15 min, and supernatant´s aliquots for each evaluation were subsequently taken. The rest of volume of complete uncentrifuged homogenates were kept at −80 °C until further use.
For antioxidant enzymes activity determination, all of absorbance was measured in a Shimadzu Double-Beam Scanning UV–Vis Spectrophotometer (Model UV-1700, SHIMADZU Corporation, Nishinokyo Kuwabara-cho, Nakagyo-ku, Kyoto 604-8511, Japan). All chemical reagents used were obtained from Sigma–Aldrich Company (St. Louis, MO, USA), unless otherwise indicated. Previous techniques described for catalase (CAT) [64 (link)], superoxide dismutase (SOD) [65 (link)] and glutathione peroxidase (GSH-Px) [66 ] were followed. Finally, Malonaldehyde (MDA), one of the end products of lipid peroxidation (LPO) reaction, was quantified in renal tissue also using Thiobarbituric acid reactive substances (TBARS) technique described by Ohkawa et al. [67 (link)].
For antioxidant enzymes activity determination, all of absorbance was measured in a Shimadzu Double-Beam Scanning UV–Vis Spectrophotometer (Model UV-1700, SHIMADZU Corporation, Nishinokyo Kuwabara-cho, Nakagyo-ku, Kyoto 604-8511, Japan). All chemical reagents used were obtained from Sigma–Aldrich Company (St. Louis, MO, USA), unless otherwise indicated. Previous techniques described for catalase (CAT) [64 (link)], superoxide dismutase (SOD) [65 (link)] and glutathione peroxidase (GSH-Px) [66 ] were followed. Finally, Malonaldehyde (MDA), one of the end products of lipid peroxidation (LPO) reaction, was quantified in renal tissue also using Thiobarbituric acid reactive substances (TBARS) technique described by Ohkawa et al. [67 (link)].
4
Quantifying Gut-Associated Cytokines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cytokines IL-10, IFN-γ, and TNF-α were quantified in serum and jejunum samples by using Simplex Luminex kits for each immune parameter and ProcartaPlex Basic Rat kits (eBioscience, Vienna, Austria) in a Luminex 100 IS™ (Luminex Corporation. Austin, TX, USA). The jejunum samples (100 mg) were first manually disaggregated with sterile scalpels, and the resulting tissue sections were homogenized in 0.5 mL RIPA buffer containing a cocktail of protease inhibitors (Complete, Mini tablets, Roche Life Science, Mannheim, Germany). Homogenization was completed by using a TissueRuptor® device (Qiagen, Hilden, Germany), and the samples were kept on ice during the procedure. Then, the samples were centrifuged (16,000× g for 10 min at 4 °C), and the protein concentration of the resulting supernatant was quantified and adjusted to 10 mg/mL for cytokine assessment as recommended by the manufacturer. These measurements were carried out in duplicate for each sample.
5
Western Blot Analysis of BDNF and TrkB
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CA1 and CA3 tissue were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, pH 7.4, 1% NP-40, 0.1% SDS, 0.5% Na deoxycholate, 4 mM EDTA, 150 mM NaCl, protease and phosphatase inhibitors [cOmplete mini tablets (Roche)], 10 mM sodium pyrophosphate, 50 mM NaF, and 2 mM sodium orthovanadate). Total protein concentration was quantified by BCA (bicinchoninic acid) protein assay. Twenty micrograms of total protein per well was loaded on SDS-PAGE gels and transferred to nitrocellulose blots, and then placed in blocking solution for 60 min at room temperature. Blots were incubated in anti-BDNF (1:2,000; Abcam), anti-TrkB (1:2,500; Abcam), or anti-GAPDH (1:50,000; Cell Signaling) antibodies at 4°C overnight. HRP-conjugated anti-mouse secondary antibody was used for BDNF (1:2,000), and anti-rabbit secondary antibody for TrkB (1:2,000) and GAPDH (1:10,000). Bands were developed with enzymatic chemiluminescence (ECL) and detected by UVP Biospectrum imaging system. Image files were analyzed with ImageJ. BDNF or TrkB signals were normalized to their respective GAPDH signals and expressed as percentage to the control group.
6
ELF3 Protein Stability Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
WT and XBAT31ox-1 plants grown at 22°C under long days (LDs) for 4 days were transferred to 29°C for 3 days and sampled at ZT 8 hours. Seedlings were harvested, and total proteins were extracted using the extraction buffer [50 mM tris-MES (pH 8.0), 0.5 mM sucrose, 1 mM MgCl2, 10 mM EDTA, and 5 mM DTT] with freshly added protease inhibitor cocktail cOmplete Mini tablets (Roche, Shanghai, China). After that, the protein mixtures were incubated with 10 mM ATP and 50 μM cycloheximide (CHX) at 29°C for 0 to 100 min in the presence or absence of 50 μM MG132. After SDS-PAGE, the abundance of ELF3 was detected by Western blotting. Three independent experiments were performed, and each immunoblot was quantified using ImageJ software.
7
Protein Extraction and Biotinylation Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cell pellets were resuspended in a cold extraction buffer (0.5% CHAPS; 2 mM β-mercaptoethanol; 25 mM Tris-HCl; 100 mM NaCl; 20 mM CaCl2; 1 mM phenylmethylsulfonyl fluoride), with addition of protease inhibitors (Complete Mini Tablets, Roche), and disrupted with a dounce homogenizer and zirconium beads, for 20 cycles in an ice bath, to obtain the total cell extract. The preparations were centrifuged at 18,000xg for 30 min at 4°C to obtain the clarified total extracts. The protein concentrations in both extracts were determined by the Pierce™ Bicinchonic Acid (BCA) Protein Assay Kit, ThermoFisher, following manufacturer’s instruction. To analyze the protein pattern of the extracts, 10 μg of proteins were resolved by 10% SDS-PAGE, as described elsewhere (Gonçalves et al., 2019 (link)). The efficiency of surface protein biotinylation was analyzed by Western blot using an alkaline phosphatase-streptavidin conjugate (SouthernBiotech, AL, USA) and developed with an NBT/BCIP substrate (ThermoFisher) as described (Gonçalves et al., 2019 (link)).
8
Decellularization and Protein Extraction of Whole Left Ventricles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole LVs were decellularized as previously reported [23 (link)]. In summary, the tissue was incubated in decellularization buffer (1% sodium dodecyl sulfate in PBS) with 1x protease inhibitors cocktail (PI; cOmplete Mini tablets, Roche). Samples were left at room temperature in an orbital shaker until tissue was completely decellularized (three to four days). The decellularization buffer was decanted daily and replaced with fresh decellularization buffer. When tissue looked translucent, samples were considered decellularized. Tissue was washed three times in distilled water with 1x PI for 5 min and then left in fresh 1x PI/water overnight to remove all remnants of the decellularization buffer. The decellularized LVs were homogenized (Power Gen 1000, Fisher Scientific) in Protein Extraction Reagent Type 4 (7.0 M urea, 2.0 M thiourea, 40 mM Trizma base, and 1.0% C7BzO, pH 10.4) and 1x PI. Protein quantification was performed using a Coomassie Brilliant Blue G-250-based assay (Quick Start Bradford Protein Assay, Bio-Rad). All samples were stored at −80°C until use.
9
Optimized Western Blotting Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For Western blotting, equal number of cells were seeded in 6 well dishes and the samples were extracted by directly boiling with 2× Laemmli sample buffer. In certain cases, cells were lysed with 50 mM Tris (pH 7.4), 150 mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40 and protease inhibitors (Roche, complete mini tablets) at 4oC for 30 minutes, followed by centrifugation at 14000 rpm at 4oC. Proteins were separated using SDS PAGE, and then transferred to 0.2 or 0.45 μM nitrocellulose membranes. The membranes were blocked either with 5% BSA or 5% milk in 1X TBST. Primary antibodies were incubated overnight at 4oC, washed 3× with 1X TBST and then incubated with Odyssey secondary antibodies (1:10,000) for 90 minutes at room temperature. The blots were subsequently washed 2× with TBST and once with TBS and developed with an Odyssey infrared scanner.
10
Lysosomal Fraction Isolation and GCase Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To isolate the lysosomal fraction, cell pellets, collected as earlier described, were resuspended in PBS with protease inhibitor (Complete Mini Tablets, Roche) and homogenized with a 5 ml syringe with a 21 G needle. This was followed by centrifugation (1,000 g, 10 min, 4 °C) to pellet nuclei and cell debris. The supernatant was collected and centrifuged (20,000 g, 30 min, 4 °C) to pellet the lysosomal fraction. Total cell pellets or lysosomal fraction pellets were sonicated at 10 amp for 10 sec in citrate phosphate buffer pH 5.4 consisting of 0.1 M citric acid (Sigma) and 0.2 M dibasic sodium phosphate (Sigma) with 0.25% (v/v) Triton X and 0.25% (w/v) taurocholic acid (Sigma). Samples were centrifuged at 800 g for 5 min at 4 °C, and the supernatant was collected. Then samples were diluted in citrate phosphate buffer in quadruplicate. One replicate of each sample was treated with 1 mM conduritol B epoxide (CBE, Calbiochem) for 10 min before all samples were incubated for 1 h with 2.5 mM of the fluorescent GCase substrate methylumbillifery β-D-glucopyranoside (4MUG, Sigma) at 3 °C in the dark. The reaction was quenched with 1 M glycine buffer (Sigma) pH 10.8, and the fluorescent levels were analysed on the PHERAStar FSX plate reader (BMG Labtech). Values from CBE-treated wells were subtracted as background.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!