Tissue from rat ventral midbrain was homogenized in RIPA buffer containing protease inhibitor cocktail (P8340, Sigma) and phenylmethylsulfonyl fluoride (P7626, Sigma). The homogenates were centrifuged at 12,000×g for 20 min at 4 °C, and the protein concentrations were determined by the Bradford protein assay. The levels of TNF-α were quantified with rat specific enzyme-linked immunosorbent assay kits according to the manufacturers’ instructions (rat TNF-α from Diaclone 865.000.96, Gen-Probe Diaclone SAS, Besançon, France). Finally, TNF-α content was obtained in picograms per milliliter protein and expressed as percentages of the content in the control group samples.
P7626
Manufactured by Merck Group
Sourced in United States, Spain, Germany
P7626 is a laboratory equipment product from Merck Group. It is a precision instrument designed for controlled environmental conditions.
Automatically generated - may contain errors
Lab products found in correlation
P5726, by Merck Group (9 mentions)
P8340, by Merck Group (6 mentions)
Luminata classico, by Merck Group (5 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (5 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Precision plus protein kaleidoscope standard, by Bio-Rad (4 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Bradford assay dye reagent, by Bio-Rad (3 mentions)
Phosphatase inhibitor, by Merck Group (3 mentions)
Protease inhibitor, by Merck Group (3 mentions)
Proteinase k, by Thermo Fisher Scientific (3 mentions)
Iseq00010, by Merck Group (2 mentions)
G8795, by Merck Group (2 mentions)
P0044, by Merck Group (2 mentions)
Immobilon p polyvinylidene fluoride pvdf transfer membrane, by Merck Group (2 mentions)
Luminata crescendo western horseradish peroxidase hpr substrates, by Merck Group (2 mentions)
M6250, by Merck Group (2 mentions)
A1046, by ITW Reagents (2 mentions)
A3559, by ITW Reagents (2 mentions)
Nah2po4, by ITW Reagents (2 mentions)
47 protocols using p7626
1
Quantifying Rat Midbrain TNF-α Levels
2
Spd2 Protein Expression Analysis in Larval Brains
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Twenty third instar larval brains of each genotype were dissected in cold PBS supplied with 1% protease inhibitor cocktail (P8340, Sigma) and 1mM PMSF (P7626, Sigma) and collected in a tube. 20 ml of sample buffer was added to the brains and the tissues were stripped with the help of blunt forceps on ice to induce mechanical dissociation. Samples were boiled at 70°C for 10min. Samples were run in 10% Bis/Tris gel in MOPSSDS buffer at 180V (Np0301 and NP0001 from NuPAGE-ThermoFIsher) and transferred fir 1h at 100V in the cold using 0.2 μm NC nitrocellulose (GE Healthcare Life Science). Membranes were then blocked in PBS supplemented with 0.1% Tween20 (PBST) and 10% dried milk powder for 30 min followed by incubation O/N 4˚C with Spd2 primary antibody at 1:500 (Dix and Raff, 2007 (link)) , diluted in PBST with 3% dried milk powder. Membranes were washed 4 times for 10min in PBST and then incubated for 2h at room-temperature with a Rabbit secondary antibody conjugated to Horseradish peroxidase (HRP) (# G21234, Life Technologies) diluted in PBST with 3% dried milk powder. The secondary antibody solution was then removed and membranes washed 5 times for 10min in PBST. Finally, membranes were incubated with SuperSignalTM West Pico Chemiluminescent Substrate (34080, Termo ScientificTM) and revealed using the BioRad Chemidoc MP system. Images were analyzed using ImageLab software.
3
Visualizing Autophagy in Fusarium oxysporum
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Micro conidia (2.5 × 108) inoculated on PDA medium were grown for 15 h at 28 °C to visualize the autophagy in the F. oxysporum. The strains were sifted to an SM medium without a N source after washing with sterile water in the presence of 4 mM PMSF (P7626, Sigma-Aldrich, St. Louis, MO, USA). The mycelia were stained in the dark for 30 min with the fluorescent dye MDC (monodansylcadaverine) with concentration of 50 mM, (Sigma, D4008) after 1 h starvation followed by washing with water and observation under epifluorescence and differential interference contrast (DIC) microscopy [26 (link)].
4
ROCK1/2 Expression in HUVECs under GHS Conditions
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The HUVECs on Flat, HP1, HP2, and HP3 GHS for 2 days were washed twice with PBS and disrupted with 1 × cell lysis buffer (9803, Cell Signaling Technology) containing 1 mM phenylmethylsulfonyl fluoride (P7626, Sigma). A quantitative analysis of the samples was performed using the Bradford assay dye reagent (500–0006, Bio-Rad). The sample protein (10 μg) was boiled in 1 × loading dye for 5 min and subjected to electrophoresis in a 10% polyacrylamide gel with sodium dodecyl sulfate. After transfer to a polyvinylidene fluoride membrane (ISEQ. 00010, Millipore), the membranes were blocked with 5% bovine serum albumin containing 1 × TBST (a mixture of Tris-buffered saline and Tween 20; WH400028806, 3 M) at RT for 1 h. The membranes were incubated with anti-ROCK1 (1:1000; ab45171, Abcam), anti ROCK2 (1:1000; ab71598, Abcam), and anti-GAPDH (1:2000; G8795, Sigma) antibodies at RT for 2 h. The membranes were then washed three times with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody (1:3000; Santa Cruz) in TBST at RT for 1 h. Chemiluminescence was visualized with the ECL Plus reagent (32132, Thermo Fisher Scientific) and recorded on X-ray film.
5
Drosophila Protein Extraction and Immunoblotting
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For each genotype, 15 brains from third-instar larvae were dissected in PBS, 1% protease inhibitor cocktail and extracted in 100 μl of cold protein extract buffer (50 mM trisHCl pH 7.4 (200923A, Euromedex), 50 mM NaCl (27810, Prolabo), 1 mM EDTA (E9884, Sigma), 0.25% DOC (D6750, Sigma), 0.1% SDS (EU0660, Euromedex), 1 mM PMSF (P7626, Sigma), 1‰, DTT (D0632), 1% of protease inhibitor solution (1 mM benzamidine-HCl (B6506, Sigma), 0,1 mg ml−1 phenanthroline (131377, Sigma), 1 mg ml−1 aprotinin (A6103, Sigma), 1 mg ml−1 leupeptin (L2884, Sigma), 1 mg ml−1 pepstatin A (P5318, Sigma)). Protein extracts were separated by SDS–polyacrylamide gel electrophoresis with either a 10% BIS-TRIS or a 3-8% TRIS-Glycine NuPAGE gel (Invitrogen) and then transferred on a nitrocellulose membrane (Protran BA83, Whatman). Membranes were probed with antibodies anti-Fzy (1:1,000, a gift from J. Raff lab) and anti-α-tubulin (DM1Α) (1:1,000, Sigma-Aldrich).
6
Quantifying Protein Expression in Corpus Luteum
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Fresh CL tissue (early CL, n = 6; mid CL, n = 6; late CL, n = 6) and in vitro tissue explants (mid CL, n = 6) were disrupted by homogenization in RIPA (250 μL) containing protease inhibitor (P8340, Sigma-Aldrich), phospho-stop solution (88667, ThermoFisher) and phenylmethylsulfonyl fluoride (PMSF) (P7626, Sigma-Aldrich) at 4°C. Protein concentration was determined with bicinchoninic acid assay (BCA) (BCA1-1KT, Sigma-Aldrich). A total of 10–80 μg of protein was run on 6–12% (varying accordingly to each protein) polyacrylamide gel followed by transfer to nitrocellulose membranes. Then, membranes were incubated with primary antibodies (Table 2 ) at 4°C, overnight. Goat anti-mouse alkaline phosphatase conjugated antibodies (1:30,000, 31321, ThermoFisher), goat anti-rabbit alkaline phosphatase-conjugated antibodies (1:30,000, A3687, Sigma-Aldrich), and rabbit anti-goat alkaline phosphatase-conjugated antibodies (1:30,000, A4187, Sigma-Aldrich) were used as a secondary antibody. Immune complexes were visualized using alkaline phosphatase substrate. Blots were scanned in Molecular Imager VersaDoc MP 4000 System (BioRad, Hercules, California, USA) and specific bands quantified using ImageLab Software (BioRad). At last, band density for each of the target protein was normalized against β-actin.
7
Parasite Homogenization and Protein Quantification
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The CWAs were prepared according to a protocol previously described by Adisakwattana et al. [10 (link)]. The parasite specimen was homogenized with lysis buffer containing 0.01 M phosphate-buffered saline (PBS, pH 7.2), 10 mM Tris-HCl, 150 mM NaCl, 0.5% Triton X-100, 10 mM EDTA, and 1 mM PMSF (P-7626, Sigma-Aldrich, Inc.) using a glass tissue homogenizer. The mixture was then sonicated at 20% amplitude for 5 min in an ice bath with a 15 s pulse on and 15 s pulse off, using an ultrasonic processor, the Vibra cell™ VC-505 (Sonics & Materials Inc., Newtown, USA). Subsequently, the suspension was centrifuged at 5000 g for 20 min at 4°C to remove insoluble materials. Finally, the supernatant was collected and measured for protein concentration using the Bradford protein assay [11 ] with Bio-Rad protein assay reagent kits (Bio-Rad Laboratories Inc., Hercules, USA). Bovine serum albumin was used as a standard.
8
Neuronal Protein Extraction for Western Blot
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Based on previous in vitro experiments described [26 (link), 27 (link)], control and mutated neurons were treated with D-JNKI1 at 2 μM concentration starting on day 28 of terminal differentiation. On day 30, neurons were isolated as described above to obtain proteins for Western blot analysis. Proteins were extracted with sucrose 1.09 g (S0389, Sigma-Aldrich, Darmstadt, Germany), NaHCO3 1 mM (S5761, Sigma-Aldrich Darmstadt, Germany), MgCl2 1mM (M8266, Sigma-Aldrich Darmstadt, Germany), Hepes 1 mM (H3375, Sigma-Aldrich, Darmstadt, Germany), NaF 10 mM (201154, Sigma-Aldrich, Darmstadt, Germany) Triton X-100 0.1% (X100, Sigma-Aldrich, Darmstadt, Germany), DTT 1mM (GE17-1318-01, Sigma-Aldrich, Darmstadt, Germany), NaOH 1 mM (S8045, Sigma-Aldrich, Darmstadt, Germany), PMSF 1 mM (P7626, Sigma-Aldrich, Darmstadt, Germany), and protease inhibitor (4693124001, 04906837001, Complete; Roche Diagnostics, Basel, Switzerland).
9
Fecal Sample Preparation for ELISA
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Fecal sample preparation of enzyme-linked immunosorbent assay (ELISA) has been previously described (33 (link)). One hundred milligrams of fecal pellets were homogenized in 3 ml of collection media consisting of 0.05 mg soybean trypsin inhibitor per ml of a 3:1 mixture of 1× PBS and 0.1 m EDTA, pH 7.4. Following centrifugation at 1800 rpm for 10 min, the supernatant was centrifuged again at 14,000 rpm for 15 min at 4 °C, and final supernatant was collected and stored with 20% glycerol and 2 mm phenylmethylsulfonyl fluoride (Sigma, P-7626) at −20 °C until analysis.
10
Brush Border Membrane Enzyme Assay
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After 3–4 days of culture on conventional 24-well plates, RTgutGC cells were exposed to LPS and the functional ingredients for 6 h. After discarding the mucosal saline with LPS or functional ingredients, cells were harvested by trypsination and centrifugation. Cell pellets were reconstituted in 1 mL ice-cold 2 mM Tris/50 mM mannitol pH 7.1, containing phenyl-methyl-sulphonyl fluoride (P-7626, Sigma, Norway) as serine protease inhibitor. Brush border membrane enzyme activities, i.e., leucine amino peptidase (LAP) and maltase, were subsequently measured. LAP activity was analyzed colorimetrically with a commercial kit (NO. 251, Sigma, Norway) using L-leucine-β-naphthylamide as substrate according to the methods described by Krogdahl et al. (27 (link)). Maltase activity was measured using maltose as substrate according to the description of Dahlquist (28 (link)). Total protein concentrations were determined using a Bio-Rad Protein Assay (Bio-Rad Laboratories, Munich, Germany). Enzyme activities were expressed as mol substrate hydrolysed per hour per mg protein.
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