Harvested viruses were inactivated with 0.01% formaldehyde at 37 °C for overnight. The inactivated viruses were purified by using ultracentrifuge (100,000× g, 4 °C, 1.5 h with 20% sucrose as a cushion. Protein content was measured by Lowry assay (Pierce™ Modified Lowry protein assay kit, Thermo Fisher Scientific) and HA contents were measured by using single radial immunodiffusion (SRID) [12 (link)] with the WHO standard reagents. If the WHO standard reagents were not available, HA content was measured by using densitometry of SDS-PAGE [13 (link)].
Pierce modified lowry protein assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce™ Modified Lowry Protein Assay Kit is a colorimetric method for the determination of protein concentration in aqueous solutions. The kit includes reagents and instructions for performing the assay.
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Lab products found in correlation
Spectramax i3, by Molecular Devices (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Benchmark plus microplate spectrophotometer system, by Bio-Rad (1 mentions)
Costar 96 well plate, by Corning (1 mentions)
Hybridoma sfm, by Thermo Fisher Scientific (1 mentions)
Hiprep 26 10 desalting, by GE Healthcare (1 mentions)
Amicon, by Merck Group (1 mentions)
Vivaflow 200, by Sartorius (1 mentions)
Collagenase, by Merck Group (1 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Toxinsensor chromogenic lal endotoxin assay kit, by GenScript (1 mentions)
Trypsin, by Merck Group (1 mentions)
Spectramax m3, by Molecular Devices (1 mentions)
Model dr 3900, by HACH (1 mentions)
Odyssey, by LI COR (1 mentions)
Creatine kinase activity assay kit mak116, by Merck Group (1 mentions)
18 protocols using pierce modified lowry protein assay kit
1
Viral Inactivation and Purification
2
Quantifying Total Protein in ECM
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Total protein content was quantified with a Pierce™ Modified Lowry Protein Assay Kit (Thermo Fisher Scientific). For this, 2 µL of pre-gel solution was diluted in 38 µL of 1x DPBS and transferred to a well in a non-adhesive Costar® 96-well plate (Corning Inc., Kennebunk, ME, USA). Next, 200 µL of modified Lowry protein assay was added per well before incubation at RT for 10 min. Fresh 1 N Folin-Ciocalteu’s phenol reagent was prepared by diluting 2 N Folin-Ciocalteu’s phenol reagent with an equal volume of Milli-Q® water and 20 µL of this solution was added per well and incubated at RT for 30 min. The absorbance was read at 750 nm with Benchmark Plus™ microplate spectrophotometer system (Bio-Rad, Hercules, CA, USA). The protein concentration was determined based on a calibration curve derived from a dilution series of bovine serum albumin (Thermo Fisher Scientific). DPBS served as absorbance blanks. The protein concentration (µg/mL) from each organ-derived ECM was calculated from four independent experiments each performed in triplicate (Figure 1 d).
3
Purification of Anti-STn IgM Antibodies
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For antibody purification, L2A5 hybridoma cells were cultured in serum-free medium (Hybridoma-SFM, Thermo Scientific, 12045076). Hybridoma supernatant containing L2A5 mAbs was recovered and centrifuged at 1000 g for 10 min. The supernatant was collected and diluted twofold in PBS. Considering the composition of the Hybridoma-SFM medium used, low level of contaminants was present in the recovered supernatant and therefore a desalting strategy was considered useful to recover and purify the anti-STn IgM antibodies. For that, the supernatant was loaded onto desalting columns (HiPrep 26/10 Desalting, GE Healthcare Life Sciences, 45-000-266) and dialyzed against PBS (pH 7.2). Due to the high volume of supernatant available, four desalting columns were connected and used in series. The protein peak was registered by spectrophotometry measuring the OD at 280 nm. Eluted fractions containing antibodies were further concentrated using a 200 kDa MW cut-off concentrator system (Vivaflow® 200, Sartorius). A final concentration step to reduce the final sample volume was performed by ultrafiltration using a 100 kDa MW cut-off filter (Amicon, Millipore). Antibody concentration was assessed using the Pierce™ modified Lowry protein assay kit (Thermo Scientific, 23240)59 .
4
Protein Expression Analysis of Tumouroids
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Cells were extracted from tumouroids using 500 units/ml collagenase (Sigma Aldrich) for 1 hour at 37 °C. Cells in 2D and 3D were lysed in RIPA buffer containing protease inhibitors (Sigma) and quantified using the Pierce™ Modified Lowry Protein Assay Kit (Thermo Scientific) according to the manufacturers protocol. Equal amounts of protein (35 μg) were loaded in gels (BioRad), resolved by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked for 30 minutes in 2.5% BSA and 0.05% Tween 20 in PBS. Antigens were detected using mouse monoclonal antibodies against MMP-7 and vimentin (both at 1:1,000, Santa-Cruz Biotechnology, Santa Cruz, CA) and incubated with a goat anti-mouse IgG-HRP (Santa Cruz) secondary antibody at room temperature for 30 minutes. Blots were developed using the Clarity™ Western ECL Substrate (Bio-Rad, Hertfordshire) and visualized using the ChemiDoc™ XRS+ System (Bio-Rad).
5
Oxidation of Isolated LDL
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LDL was isolated and prepared as previously described [7 (link)]. LDL was isolated by ultracentrifugation (2 × 24 h, 59,000 rpm at 4 °C) with potassium bromide density adjustments (0.01906 and 0.06583 g/mL KBr/plasma). LDL was dialyzed against PBS (4× against 1 × PBS, pH 7.4 at 4 °C for 1 h, 2 h, 3 h, and overnight) to remove residual potassium bromide. Then, LDL was oxidized using 20 µmol CuSO4/L (for 24 h at 37 °C) followed by further dialysis and sterile filtration (pore diameter 0.45 µm). Protein concentrations were measured with Pierce Modified Lowry Protein Assay Kit (ThermoFisher, Rockford, IL, USA, #23,240) according to the manufacturer’s instructions. Endotoxin contamination was tested (<0.01 EU/mL) by ToxinSensor chromogenic LAL endotoxin assay kit (GenScript, Piscataway, NJ, USA, #ABIN491527) according to the manufacturer’s instructions.
6
Bioluminescent Assay of Platelet Dehydrogenases
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Bioluminescent method was used to determine the levels of activity of NAD- and NADP-dependent dehydrogenases in platelets [43 (link)]. The activity of the following enzymes was determined: glucose-6-phosphate dehydrogenase (Glu6FDH), glycerol-3-phosphate dehydrogenase (Gly3PDH), NADP-dependent malate dehydrogenase decarboxylated (NADP–MDH), NAD-dependent reaction (LDH) and NADH-dependent reaction of lactate dehydrogenase (NADH–LDH), malate dehydrogenase, NAD-dependent reaction (MDH) and NADH-dependent reaction of malate dehydrogenase (NADH–MDH), NADP-dependent glutamate dehydrogenase (NADP–GluDH) and NADPH-dependent glutamate dehydrogenase (NADPH–GluDH), NAD-dependent glutamate dehydrogenase (NAD–GluDH) and NADH-dependent reaction of glutamate dehydrogenase (NADH–GluDH), NAD-dependent isocitrate dehydrogenase (NAD–ICDH) and NADP-dependent isocitrate dehydrogenase (NADP–ICDH) and glutathione reductase (GR).
The activity of NAD- and NADP-dependent dehydrogenases was expressed in enzymatic units on 1 mg of protein (1 U = 1 µmol/min) [17 (link)].
Protein concentration was determined using Pierce™ modified Lowry protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA).
The activity of NAD- and NADP-dependent dehydrogenases was expressed in enzymatic units on 1 mg of protein (1 U = 1 µmol/min) [17 (link)].
Protein concentration was determined using Pierce™ modified Lowry protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA).
7
Exosome Isolation from Mice and Cell Culture
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The plasma obtained from control or 4T1 tumor-bearing mice was centrifuged for 1000 g for 10 min. The resulting supernatant was further diluted in PBS and ultra-centrifuged (Beckman Coulter Optima LE-8OK, 70 Ti rotor) at 100,000 g for 1 h at 4 °C. The samples were washed once with PBS and centrifuged again. The pellet was resuspended in ice cold PBS, filtered with a 0.2 μm filter and kept at −20 °C until analysis. Exosomes were isolated from the cell culture medium using ExoQuick-TC™ according to the manufacturer’s instructions. The supernatant obtained from 4T1 cells cultured for 48 h in serum free media, was centrifuged at 3000 g for 15 min to remove debris and then incubated overnight at 4 °C with ExoQuick-TC™. This mixture was sequentially centrifuged for 15 and 5 min at 1500 g. The supernatant was removed and the pellet was resuspended in ice cold filtered PBS and stored at −20 °C for no more than one week. Exosome protein content was measured using the Pierce Modified Lowry Protein Assay Kit (Thermo Fisher Scientific) according to manufacturer’s instructions.
8
HDAC Activity Assay Protocol
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Cells were extracted with HDAC reaction buffer (50 mM Tris HCl pH = 7.5, 5% glycerol, 0.3% Triton-100, 50 mM NaCl) and frozen at −80 °C for later use. Thawed samples were centrifuged at 20,000× g for 15 min at +4 °C and protein concentration was determined with Pierce™ Modified Lowry Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). For each HDAC activity reaction, 25–50 µg of protein lysate was mixed with 30 µM of HDAC activity substrate (S)-tert-Butyl (6-acetamido-1-((4-methyl-2-oxo-2H-chromen-7-yl)amino)-1-oxohexan-2-yl)carbamate (Boc-Lys(Ac)-AMC) (Sigma Aldrich, St. Louis, MO, USA) in the reaction buffer (50 mM Tris HCl pH = 8, 100 mM NaCl) and incubated for 30 min at 30 °C. The reaction was stopped with 10 mg/mL trypsin and 25 µM SAHA (Sigma Aldrich, St. Louis, MO USA). Fluorescence was measured at 325/395 nm using spectrophotometer SpectraMax i3 (Molecular Devices, San Jose, CA, USA).
9
Determination of Algae Protein Solubility
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The determination of solubility of the extracted DNOB isolates was based on Zhao et al. [21] (link) where 1 % (w/v) algae solution was prepared by dissolving the extracted protein isolates (Control, HPH2P, HPH3P, US20 and US40) in 10 mM sodium acetate buffer, followed by being kept at 4 °C overnight to allow full hydration. Then 2 M HCl or 2 M NaOH was used to adjust the protein suspension to pH 2 – 12 and protein samples at each pH were centrifuged at 8000 × g for 15 min at 4 °C. The solubilized protein content in collected supernatants was determined by Pierce™ Modified Lowry Protein Assay Kit (Thermo Scientific™, Rockford, USA) using bovine serum albumin as calibration standard. Additionally, the total protein content was determined using Kjeldahl method mentioned in section 2.3 . The solubility of the algae was calculated as followed in equation (1) .
10
ATP Quantification in Human Mesenchymal Stem Cells
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Cells (3 × 103/well) were seeded in a white, clear-bottom, 96-well plate. ATP measurements were done by ATPlite 1step Luminescence Assay, adding 100 µL of ATPlite 1step solution to each well as recommended by manufacturers (PerkinElmer, Waltham, MA, USA). Luminescence was measured immediately with SpectraMax i3 (Molecular Devices, San Jose, CA, USA) spectrophotometer. ATPlite 1step Luminescence Assay System buffer immediately lysed cells to evaluate the total ATP amount. Before the measurement, samples were centrifuged at 20,000× g for 15 min at +4 °C. For ATP measurements in healthy and pathological hmMSC, luminescence was normalized to the cell number (pM of ATP per cell) using ATP standard curve. ATP level in the cells incubated for 3, 7, and 14 days with SAHA was normalized to protein level by lysing cells with lysis buffer (50 mM Tris HCl pH = 6.8, 10% glycerol, 1% SDS) on a separate 96-well plate and measuring with Pierce™ Modified Lowry Protein Assay Kit, (Thermo Fisher Scientific, Waltham, MA, USA).
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