HL60/ADR, Kasumi-1 cells were treated with 1.0, 2.0, 4.0μM decitabine for 72h. Cells were harvested and lysed in cold lysis buffer (0.5 M Tris-HCl, pH 6.8, 2 mM EDTA, 10% glycerol, 2% SDS, 5% β-ercaptoethanol) containing 0.5mM dithiothreitol (DTT) and 0.1mM phenylmethylsulfonyl fluoride (PMSF), the supernatant was collected after centrifugation (12000rpm, 10min). The protein concentration was determined using BCA protein determination kit (Pierce Chemical, Rockford, IL, USA). Cell extracts were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% milk in Tris-buffered saline containing 0.1% Tween 20, and immunoblotted overnight at 4°C with the appropriate primary antibody, followed by treatment with horseradish peroxidase-linked secondary antibody. The immunocomplex was visualized using a chemiluminescence phototope-horseradish peroxidase kit. Antibody against β-actin (CST, MA, USA) was used to ensure equivalent loading of whole cell protein.
Bca protein determination kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The BCA protein determination kit is a colorimetric assay used for the quantitative determination of total protein concentration in a solution. The kit utilizes the bicinchoninic acid (BCA) reaction, where proteins reduce Cu2+ ions to Cu+ ions in an alkaline environment. The resulting purple-colored reaction is measured spectrophotometrically, and the protein concentration is determined by comparison to a standard curve.
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Lab products found in correlation
Ripa lysis buffer, by Beyotime (3 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (3 mentions)
Complete protease inhibitor, by Merck Group (2 mentions)
Anti total stat3, by Abcam (2 mentions)
Anti il 6, by Abcam (2 mentions)
Sonic dismembrator model 100, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence, by Merck Group (1 mentions)
Veriti 96 well thermal cycler, by Thermo Fisher Scientific (1 mentions)
β tubulin sc 9104, by Santa Cruz Biotechnology (1 mentions)
Anti phospho stat3 tyr705, by Cell Signaling Technology (1 mentions)
Goat anti rabbit secondary antibody, by R&D Systems (1 mentions)
Anti crp, by Abcam (1 mentions)
Anti phosho akt ser473, by Cell Signaling Technology (1 mentions)
Anti total erk, by Cell Signaling Technology (1 mentions)
Anti phospho stat3 tyr705, by Abcam (1 mentions)
Clear 96 well plate, by BD (1 mentions)
White 96 well plate, by Thermo Fisher Scientific (1 mentions)
Atp determination kit, by Thermo Fisher Scientific (1 mentions)
Flexstation 3, by Molecular Devices (1 mentions)
Peroxidase affinipure goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
18 protocols using bca protein determination kit
1
Decitabine Cytotoxicity in Leukemia Cell Lines
2
Western Blot Analysis of NVP-LDE225 Effects
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Western blot analysis was carried out as described previously.3 (link) Briefly, HL60/ADR and primary AML cells were cultured for 48 h with different concentrations of NVP-LDE225. Cells were harvested and lysed in 200 µL cold lysis buffer (0.5 M Tris-HCl, pH 6.8, 2 mM EDTA, 10% glycerol, 2% SDS, 5% β-mercaptoethanol) containing 0.5 mM dithiothreitol (DTT) and 0.1 mM phenylmethylsulfonyl fluoride (PMSF). The supernatant was collected after centrifugation (12,000 rpm, 20 min), and protein concentration was determined using a BCA protein determination kit (Pierce Chemical, Rockford, IL, USA). Cell extracts were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% skimmed milk or 5% BSA in Tris-buffered saline containing 0.1% Tween 20 and then incubated overnight at 4 °C with appropriate primary antibodies, followed by a horseradish peroxidase-linked secondary antibody. Reactions were visualized by enhanced chemiluminescence (Millipore, USA). Antibodies specific for β-actin or GAPDH were used to ensure equivalent loading.
3
Thermal Shift Assay for CETP-Ligand Binding
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The protein-drug binding thermal shift assay was employed to test the binding of PL/AP to CETP [46 (link)]. Briefly MCF-7 cells grown to 60% confluency in T75 flasks were treated with PL, AP or Torcetrapib for 2 h at 37°C. Cells were trypsinized, counted, and washed twice with 1x PBS. The cell pellet was resuspended in 1 × EDTA-free protease inhibitors and aliquoted into PCR tubes at 1.5 × 106 cells per tube. Tubes were placed in a Veriti® 96-Well Thermal Cycler (Applied Biosystems) and incubated for 3 min at 56–74°C with 2°C increments. Tubes were taken out of the thermal cycler and incubated at room temperature for 3 min. Cells were lysed by freeze-thawing the tubes twice in liquid nitrogen and at 25°C for 3 min with vortexing in between freeze-thaw cycles. Immediately after, cells were centrifuged at 14 000 rpm and 4°C for 20 min, and the supernatant was transferred to a clean Eppendorf tube. Protein was quantified using the BCA protein determination kit (Pierce Thermo Scientific, USA), and equal amounts of protein (15-20 μg) were subjected to Western blotting for CETP detection.
4
Protein Expression Analysis in MCF-7 Cells
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Protein lysate harvested from MCF-7 cells was quantitated with the BCA protein determination kit (Pierce Thermo Scientific, USA). An equal amount (20-30 μg) of protein lysate was subjected to gel electrophoresis on 10% SDS page gels, transferred to nitrocellulose membrane and probed with antibodies to PARP-1 (C2- 10, Trevigen, diluted 1:1000), Pro-Caspase-7 (PRS3467, Sigma, diluted 1:1000), Pro-Caspase-9 (MA1-12562, Thermo Scientific, diluted 1:1000), pH2AX (ADI-KAM- CC255, Enzo Life Sciences, diluted 1:4000), and CETP (PA1-050, ABR, diluted 1:500). β-Tubulin (sc-9104, Santa Cruz Biotechnology, diluted 1:1000) was used as a loading control. Fold change in expression of the serum protein was determined using densitometry analysis of Western blots using ImageJ.
5
Protein Quantification with BCA Assay
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Protein concentrations were determined with BSA as a standard using a bicinchoninic acid (BCA) protein determination kit (Thermo Scientific; Waltham, MA, USA) according to the manufacturer’s instructions.
6
Protein Extraction and Western Blot Analysis
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Proteins from tissue (1 × 2 mm) or BON1 cells were extracted using RIPA buffer containing complete protease inhibitors (Sigma-Aldrich) according to the manufacturer’s instructions, and the protein concentration was determined using a BCA Protein Determination Kit (Thermo Scientific) following the manufacturer’s instructions. Western blots were processed as previously described (31 (link)) using the following antibodies: anti-IL-6, anti-total-STAT3, and anti-ATX (all from Abcam); and anti-phospho-STAT3 (Tyr705) (Cell Signaling Technology). A goat anti-rabbit secondary antibody was used (R&D Systems). Protein expression was quantified using ImageJ (NIH).
7
Protein Extraction and Western Blot Analysis
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Proteins from frozen pNEN tissue specimen (60–80 mg) pulverized in liquid nitrogen or from BON1 cells pellets were extracted using RIPA buffer containing complete protease inhibitors (Sigma-Aldrich) and supplemented with phosphatase inhibitor sets 1 and 2 (Sigma-Aldrich). Using an ultrasonic homogenizer (SonoPuls mini20 Bandelin®, Berlin, Germany), the suspensions were subjected to a 30-s sonication step on ice (ampl. 80%, 0.99 kJ) and subsequently centrifuged at 16,000 g for 10 min.
Supernatants were collected and divided into aliquots, and the total protein concentration was determined using a BCA Protein Determination Kit (Thermo Scientific) following the manufacturer’s instructions.
Western blot was performed as previously described (37 (link)) using the following monoclonal antibodies: anti-CRP, anti-IL-6, anti-total-STAT3 (all Abcam), anti-phospho-STAT3 (Tyr705), anti-total-ERK, anti-phospho-ERK (Thr202/Tyr204), anti-total-AKT and anti-phosho-AKT (Ser473) (all Cell Signaling). The secondary antibody used was goat anti-rabbit (R&D Systems). Quantification of protein expression was performed using ImageJ (EHD imaging, Damme, Germany).
Supernatants were collected and divided into aliquots, and the total protein concentration was determined using a BCA Protein Determination Kit (Thermo Scientific) following the manufacturer’s instructions.
Western blot was performed as previously described (37 (link)) using the following monoclonal antibodies: anti-CRP, anti-IL-6, anti-total-STAT3 (all Abcam), anti-phospho-STAT3 (Tyr705), anti-total-ERK, anti-phospho-ERK (Thr202/Tyr204), anti-total-AKT and anti-phosho-AKT (Ser473) (all Cell Signaling). The secondary antibody used was goat anti-rabbit (R&D Systems). Quantification of protein expression was performed using ImageJ (EHD imaging, Damme, Germany).
8
Evaluating Neuroprotective Effects of Compounds
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Ten thousand HT-22 cells per well were seeded into a clear 96-well plate (FALCON) one day prior to assay. Medium was removed from the plate, and cells were treated with a 25 µL solution of each compound at 10 µM and incubated at 37 °C for 10 min followed by the addition of a 25 µL of Aβ (American peptide) solution at 10 µM. Cells were incubated at 37 °C for 7 h and washed twice with PBS. Cells were lysed by using 1% Triton-X 100 in TBST buffer solution and the protein concentrations were determined by using the BCA protein determination kit (Thermo scientific). An equal amount of cell lysates from each well was plated into a white 96-well plate (NUNC) and the amount of ATP generated in each sample was determined by using the ATP determination kit (Molecular Probes, USA) containing D-luciferin and luciferase. The % inhibition value was calculated by measuring luminescence from each sample (Flexstation® 3, Molecular Devices, USA), and comparing the ATP levels of the vehicle control treated with Aβ as a negative control. Cell viability was also calculated based on the ATP levels of each sample without the treatment of Aβ solution.
9
Protein Extraction and Western Blot Analysis
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RIPA lysis buffer (#P0013B; Beyotime) was used to extract total protein from cells according to the manufacturer's protocol. Protein was quantified using a BCA protein determination kit (#23225, Thermo Fisher Scientific, USA). Following the addition of loading buffer to the protein samples, the mixture was kept in water at 100°C for 5 min and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed. Protein samples were then transferred to a polyvinylidene fluoride membrane. This membrane was sealed with 5% skim milk and blocked for 2 h at room temperature. Subsequently, it was incubated overnight at 4°C with the following primary antibodies: Bcl-2 (3498; CST), Bax (50599-2-Ig; Proteintech), cleaved caspase-3 (9664; CST), CDK2 (2546; CST), and GAPDH (60004-1-Ig; Proteintech). Peroxidase-AffiniPure goat anti-rabbit IgG (H + L; 111-035-003; Jackson) and peroxidase-AffiniPure goat anti-mouse IgG (H + L; 115-035-003; Jackson) were used as secondary antibodies. For enhanced chemiluminescence, Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) was used to detect the proteins, with GAPDH as the internal reference.
10
Melanin Quantification by Spectroscopy
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Quantification of melanin content by spectroscopy was performed essentially as described (Delevoye et al., 2009 (link)). Briefly, 1.2 × 106 cells were seeded in 6-cm dishes in triplicate. The next day, cells were rinsed twice with PBS, detached by incubation in PBS, 10 mM EDTA for 15–20, and collected by centrifugation at 500 × g for 5 min. Cells were resuspended in cold 50 mM Tris-HCl, pH 7.4, 2 mM EDTA, 150 mM NaCl, 1 mM DTT, and 1× protease inhibitor cocktail (Roche), and then sonicated on ice using the Sonic Dismembrator Model 100 (Thermo Fisher Scientific). Sonicates were fractionated into supernatant and pellet fractions by centrifugation at 20,000 × g for 15 min at 4°C. Supernatants were subjected to the determination of protein concentration by BCA protein determination kit (Thermo Fisher Scientific). Melanin-containing pellets were rinsed in ethanol/diethyl ether (1:1) and dissolved in 2 M NaOH/20% dimethyl sulfoxide at 60°C for 1 h. The melanin content of individual samples was measured by spectrophotometry as absorbance at 492 nm, and normalized to protein concentration in each sample. Triplicate samples were analyzed for each experimental condition in each experiment.
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