Cell lysates for protein expression studies were prepared as per our previous reports (29 (link), 30 (link)). Briefly, after cell harvesting, cells were lysed with Radioimmunoprecipitation assay buffer (RIPA buffer) and protein quantification of the samples was measured by the Bradford colorimetric assay (Sigma-Aldrich) using BSA as standard. Equal amounts of protein (40 µg) were loaded to polyacrylamide gel, and the proteins were resolved in 8%–15% gel using Miniprotein Tetra System (Bio-Rad). The gel was transferred to Polyvinylidene fluoride (PVDF) membranes and immunoblotted using specific antibodies against various proteins. Secondary antibodies (Horseradish peroxidase (HRP)-conjugated) at a dilution of 1:2,000 (Santa Cruz/CST-USA) and directed against primary antibodies were used, and the blots were developed with Immobilon Western Chemiluminescent HRP Substrate. Signal intensities of bands were detected and quantified by ChemiDoc Imaging System (Bio-Rad).
Bradford colorimetric assay
Manufactured by Merck Group
Sourced in United States
The Bradford colorimetric assay is a laboratory technique used to quantify the total protein concentration in a sample. It is a spectrophotometric method that relies on the binding of the Coomassie Brilliant Blue dye to proteins, resulting in a color change that can be measured and correlated to the protein concentration.
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Lab products found in correlation
Protease inhibitor, by Roche (3 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Sodium orthovanadate, by Merck Group (2 mentions)
Z36hk, by Hermle (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Mini protein tetra system, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Nonidet p 40, by Merck Group (1 mentions)
Serum bovine albumin, by Merck Group (1 mentions)
Malondialdehyde mda, by Cayman Chemical (1 mentions)
12 protocols using bradford colorimetric assay
1
Protein Expression Analysis by Western Blot
2
Western Blotting Analysis of Cancer Cell Lines
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SK-BR-3, LNCaP, and MCF-7 cells grown in 100-mm dishes at 37 °C, were collected and centrifuged at 1200 rpm for 5 min; the cell pellets were lysed in a buffer containing 10 mM Tris-HCl (pH 7.4), 0.5% Nonidet-P-40, and 150 mM NaCl in the presence of protease inhibitors (Roche, IN, USA). After incubation on ice for 20 min, the extracts were clarified by centrifugation at 12,000 rpm for 15 min at 4 °C. Protein concentration was determined by the Bradford colorimetric assay (Sigma-Aldrich), and Western blotting analyses were performed by incubating the membranes with anti-ErbB2, anti-PD-L1, or anti-EGFR antibodies, followed by the HRP-conjugated secondary antibodies. Figure S7 shows whole blots relative to the Western Blotting analyses shown in Figure 2 .
3
Western Blot Protein Quantification
4
NK Cell Cytotoxicity Assay with mAbs
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SK-BR-3 and LNCaP cells were plated at a density of 6 × 105 cells/well in six-well plates and treated for 72 h with ID-1, ID-8 or Ipilimumab mAbs (400 nM). The enriched NK cells were plated at a density of 1 × 106 cells/well in 48-well plates and treated with the above mentioned mAbs at the same concentrations for 66 h. After the treatments, cells were scraped and collected by centrifugation at 1200 rpm for 10 min. The cell pellets were resuspended in a lysis buffer of 10 mM Tris-HCl pH 7.4, 0.5% Nonidet-P-40, 150 mM NaCl, containing 1 mM Sodium orthovanadate (Sigma-Aldrich, St. Louise, MO, USA) and protease inhibitors (Roche, Indianapolis, IN, USA). After lysis, the protein concentration of cell extracts was determined by the Bradford colorimetric assay (Sigma-Aldrich, St. Louise, MO, USA) and Western blotting analyses were performed by incubating the nitrocellulose filters with the indicated commercial primary anti-phospho44/42 MAPK (indicated as pErk), anti-phospho-(Ser/Thr) Akt, anti-Cleaved Caspase-3 polyclonal antibody, anti-vinculin, anti-p-Tyr polyclonal antibodies or anti-actin antibody followed by HRP-conjugated secondary antibody used for the detection, as previously described [54 (link)].
5
Lipid Peroxidation Analysis in Renal Tissue
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For lipid peroxidation analysis, renal tissue (100 mg/mL) was homogenized in RIPA
buffer (Sigma-Aldrich) and centrifuged at 1600 g for 10 min at
4°C (Z 36 HK, Hermle-Labortechnik, Germany). Supernatant was collected to
determine thiobarbituric acid reactive substances (TBARS), as described by
Draper et al. (19 (link)). TBARS levels were
calculated based on the standard curve of malondialdehyde (MDA) (Cayman
Chemical, USA) (0 to 50 µM). TBARS levels (μM) in renal tissue were normalized
by protein concentration (mg/mL) and reported as μM/mg protein.
Peroxidase activity was measured by disappearance of H2O2at 240 nm (20 (link)), while total glutathione
peroxidase (GPx) activity was determined according to Paglia and Valentine
(21 (link)). Peroxidase and GPx activities
were normalized by protein concentration (mg/mL) and are reported as mU/mg
protein.
Total protein concentration from kidney homogenates was determined by Bradford
colorimetric assay at 595 nm (Sigma-Aldrich). Bovine serum albumin
(Sigma-Aldrich) (0 to 1.4 mg/mL) was used as standard.
buffer (Sigma-Aldrich) and centrifuged at 1600 g for 10 min at
4°C (Z 36 HK, Hermle-Labortechnik, Germany). Supernatant was collected to
determine thiobarbituric acid reactive substances (TBARS), as described by
Draper et al. (19 (link)). TBARS levels were
calculated based on the standard curve of malondialdehyde (MDA) (Cayman
Chemical, USA) (0 to 50 µM). TBARS levels (μM) in renal tissue were normalized
by protein concentration (mg/mL) and reported as μM/mg protein.
Peroxidase activity was measured by disappearance of H2O2at 240 nm (20 (link)), while total glutathione
peroxidase (GPx) activity was determined according to Paglia and Valentine
(21 (link)). Peroxidase and GPx activities
were normalized by protein concentration (mg/mL) and are reported as mU/mg
protein.
Total protein concentration from kidney homogenates was determined by Bradford
colorimetric assay at 595 nm (Sigma-Aldrich). Bovine serum albumin
(Sigma-Aldrich) (0 to 1.4 mg/mL) was used as standard.
6
Quantification of BaMV-based Protein Expression
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For quantification of TP expression levels of different BaMV-based vectors, ELISA was performed as described previously (Jiang et al., 2019 (link)). TSP samples were prepared from inoculated leaves with 1:5 (w/v) ELSIA coating buffer (0.1 M carbonate/bicarbonate buffer, pH 9.6). Following centrifugation at 12,000 × g for 10 min, the supernatant was recovered and quantified for TSP using Bradford colorimetric assay (Sigma-Aldrich, St. Louis, MO, United States). The concentration of TSP in each sample was adjusted to approximately 5.5–6.5 mg/mL and 0.8–1.0 mg/mL in IC or AWF, respectively. The 96-well microtiter plates were coated with 5-fold serial dilutions of protein extracts from non-inoculated, inoculated leaves and purified plant-made SSExt mIFNγ(SP)10 or purified mIFNγ protein derived from E. coli for standard curve and the positive control. The concentration of TP was determined by comparison with known amounts of the purified mIFNγ protein derived from E. coli. All measurements were performed in triplicates.
7
Integrin-α5 Immunoprecipitation and Western Blot
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All experimental protocols involving animals were approved by the Rutgers University IACUC Committee. Frozen, half sagittal brains were homogenized with a glass tissue grinder in lysis buffer (0.32 M sucrose, 5 mM Tris-Cl pH 6.8, 0.5 mM EDTA, 1 mM PMSF) and protease inhibitor cocktail. Samples were centrifuged at 2000 r.p.m. for 10 min and the supernatant was used for biochemical analysis. Protein content was quantified with the Bradford colorimetric assay (Sigma). One milligram of brain lysates were incubated at 4 °C overnight with 2 μg of integrin-α5 antibody. Then protein A/G agarose beads (30 μl of 50% bead slurry) were added and incubated for 2 h at 4 °C. Samples were centrifuged for 30 s at 4 °C, and the pellet was washed five times in cell lysis buffer. The pellet was resuspended with 50 μl 2 × SDS Laemmli buffer, heated at 100 °C for 10 min and centrifuged for 1 min at 14 000 × g. The sample was loaded on 8% SDS-PAGE gel and immunoblotted with either HA or Kv2.1 antibody.
8
Western Blot Protein Quantification and Detection
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For Western blot analysis, protein content, typically 40–100 μg, was quantified by the Bradford colorimetric assay (Sigma-Aldrich). Samples were dissolved in 5X sample buffer (Sodium dodecyl sulfate (SDS) 10%; Bromophenol blue 0.02%; glycerol 30%; Tris-HCL 0.5 M) and 2–5% Beta-mercaptoethanol (Sigma-Aldrich); heated at 95–100 °C for 5 min and then centrifuged at 10,000 RPM for 1 min. The proteins were resolved in 10–15% SDS-PAGE and transferred into a nitrocellulose membrane. Membranes were washed one time in Tris Buffer Saline (TBS) and then blocked in 5% solution of nonfat dry milk in Tris Buffered Saline with Tween® 20 (TBST) for 1 h. Then, membranes were stained with the primary antibody diluted at 1:1000 at 4 °C overnight. After washing 30 min with TBST, membrane were incubated with secondary antibody (Goat Anti-Rabbit IgG Antibody, (H + L) HRP conjugate or Anti-Mouse IgG antibody, dilution 1:4000) for 1 h at room temperature. Membranes were washed for 30 min in TBST and then exposed with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (ThermoFischer).
9
Immunoprecipitation of Synapsin-1 and KCNB1
10
Hippocampal Tissue Homogenization and Protein Analysis
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Approximately 100 mg of human frozen hippocampal tissue (a gift of the Harvard Brain Tissue Resource Center) or half sagittal mouse brains of either sex were homogenized with a glass tissue grinder in lysis buffer [0.32 M sucrose, 5 mM Tris-Cl pH 6.8, 0.5 mM EDTA, 1 mM PMSF, and protease inhibitor cocktail set I (Calbiochem, San Diego, CA)]. Samples were centrifuged at 2000 rpm for 10 min and the supernatant used for biochemical analysis. Protein content was quantified with the Bradford colorimetric assay (Sigma, St. Louis, MO) and dissolved in Laemmli buffer with or without reducing agents. Proteins were resolved by 8–12% SDS-PAGE and transferred to a PVDF membrane that was incubated in a 5% solution of nonfat milk in Tween 20-PBS (PBST) for 2 h at room temperature. After overnight incubation at 4 °C with the primary antibody, the membrane was washed for 20 min and incubated at room temperature with the appropriate secondary antibody.
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