1.2 × 105 MCF-7 cells per well were seeded in 24-well plates and incubated for 24 h. Then, the medium was replaced with 0.6 mL of fresh OPTI-MEM I medium containing ICD@SS@SPIONs/siGL3-Cy5 nanoparticles at a final siRNA concentration of 200 nM. After 4 or 24 h, cells were washed three times with DPBS and lysed in 100 μL lysis buffer (1% Triton X-100, 2% SDS in DPBS) at −20 °C. Seventy-five µL of lysates were transferred to quartz cuvette for fluorescence intensity measurements (λEX: 647 nm; λEM: 673 nm) on a spectrofluorophotometer (RF-5301 PC Shimadzu, Kyoto, Japan). 25 μL of lysates were used in order to determine the protein content trough the bicinchoninic acid kit for protein determination (Sigma Aldrich, Milan, Italy), according to the manufacturer’s protocol. For determination of the mean fluorescence intensity, fluorescent signals were corrected for the amount of protein in the samples. The same experiment was carried out by applying a permanent magnet (attraction force of 250 g) under the wells for the entire incubation period. Results were compared with each other and with cells treated with 200 nM siRNA in OPTI-MEM I medium. The experiment was carried out in triplicate.
Bicinchoninic acid kit for protein determination
Manufactured by Merck Group
Sourced in United States, Czechia
The Bicinchoninic Acid Kit for Protein Determination is a laboratory product used to quantify the total protein content in a sample. It is a colorimetric assay that relies on the reduction of Cu2+ to Cu+ by proteins in an alkaline medium, and the subsequent chelation of the Cu+ ions by bicinchoninic acid to produce a purple-colored complex that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Spectrafluor plus, by Tecan (2 mentions)
Msd 5972, by Agilent Technologies (2 mentions)
X ray film, by Kodak (2 mentions)
Ripa lysis buffer, by Merck Group (2 mentions)
Pierce ecl plus western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Quantity one software version 4, by Bio-Rad (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Rf 5301pc, by Shimadzu (1 mentions)
Nupage lds sample buffer 4, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Gapdh primary antibody, by Cell Signaling Technology (1 mentions)
Np 40 buffer, by Beyotime (1 mentions)
Tetrahydrofuran thf, by Merck Group (1 mentions)
Benzyl benzoate, by Merck Group (1 mentions)
Magnesium chloride hexahydrate, by Merck Group (1 mentions)
Glucose 6 phosphate dehydrogenase, by Merck Group (1 mentions)
Ethylenediaminetetraacetic acid edta, by Merck Group (1 mentions)
22 protocols using bicinchoninic acid kit for protein determination
1
Cellular Uptake of Magnetic Nanoparticles
2
Quantification of CYP1A1/CYP1A2 Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CYP1A1/CYP1A2 activity was investigated by ethoxyresorufin-O-deethylase (EROD) reaction. First, HepaRG cells were incubated for 24 h with the chosen test substances. 5 µM 3-methylcholanthrene was used as positive control. After incubation, cells were washed with PBS and then incubated for 30 min with induction medium with 2 µM ethoxy-resorufin. The concentration of resorufin in the supernatant was determined by fluorescence measurement with excitation at 535 nm and emission at 590 nm. Resorufin concentration was normalized to the protein content; therefore, protein content was measured using the Bicinchoninic Acid Kit for protein determination (Sigma-Aldrich, Munich, Germany).
3
Cell Culture Protein Extraction Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Culture media were collected and cell debris were removed by centrifugation at 1000× g for 6 min, then supernatants were stored at −80 °C. Cells were washed twice with PBS, then scraped in RIPA lysis buffer with protease inhibitor cocktail, and proteins were extracted upon incubation on ice for 30 min. Lysates were centrifugated at 21,000× g for 10 min at 4 °C to precipitate insoluble cell debris and the collected supernatants were stored at −80 °C. The protein concentration of lysates was determined using Bicinchoninic Acid Kit for Protein Determination (BCA1-1KT, Sigma-Aldrich, St. Louis, MO, USA). Cell cultured media (300 μL) were lyophilised and resuspended in 30 µL of Sample Buffer 1× (NuPAGE™ LDS Sample Buffer 4×, NP0007, Invitrogen).
4
Quantifying Intracellular Zinc in Melanoma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total and free intracellular zinc content in the explant human melanoma cultures, human melanoma cell line Bowes, and normal human melanocytes HEM were determined as described before [47 (link)]. The trypsinized and rinsed cells were dissolved in 0.35 mL 0.8% nitric acid and assayed for zinc with the inductively coupled plasma emission spectrometer MSD 5972 (Agilent Technologies, Waldbronn, Germany). Prior to analysis, aliquots of the cell samples were assayed for protein content using a BCA assay (Bicinchoninic acid kit for protein determination, Sigma-Aldrich, Prague, Czech Republic). Changes in total intracellular zinc content were expressed as µg of zinc/mg of protein.
Free intracellular zinc levels in the assayed cells were determined fluorimetrically. The cells grown in black-bottom 96-well plates were incubated with Newport Green diacetate (5 μmol in PBS, dark, 30 min at 37 °C). Fluorescence intensity was determined by the multiplate reader TECAN SpectraFluor Plus (TECAN Austria GmbH, Grödig, Austria). The results in relative light units were obtained from the raw data minus reagent blank, with changes expressed as a percentage of controls.
Free intracellular zinc levels in the assayed cells were determined fluorimetrically. The cells grown in black-bottom 96-well plates were incubated with Newport Green diacetate (5 μmol in PBS, dark, 30 min at 37 °C). Fluorescence intensity was determined by the multiplate reader TECAN SpectraFluor Plus (TECAN Austria GmbH, Grödig, Austria). The results in relative light units were obtained from the raw data minus reagent blank, with changes expressed as a percentage of controls.
5
Cell-Free Protein Degradation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cell‐free protein degradation assay was performed as described previously (Wang et al., 2009 (link)). Plants were grown at 22℃ in long‐day conditions (12 h light/12 h dark cycles) and the 3‐week‐old leaves were ground to powder in liquid nitrogen. Total proteins were extracted with a cell‐free degradation buffer (25 mmol/L Tris‐HCl pH 7.5, 10 mmol/L NaCl, 10 mmol/L MgCl2, 4 mmol/L PMSF, 5 mmol/L DTT, and 10 mmol/L adenosine triphosphate) and cell debris was removed by centrifugation at 15,000 × g for 10 min at 4℃. Total protein extracts from each of the plant materials were measured the concentration by Bicinchoninic Acid Kit for Protein Determination (Sigma Aldrich) and adjusted to equal concentrations with the degradation buffer. Then, 1,000 ng of recombinant MBP‐BES1 proteins were added in 1,000 μL plant extracts (containing 100 mg total proteins) for further reaction. The reaction mixtures were incubated at 30℃ for different periods, and 200 μL reaction mixtures were taken from the tube at each sample and analyzed by immunoblots with MBP antibody.
6
Western Blot Analysis of SOCS1, pJAK2, and pSTAT3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted with RIPA Lysis Buffer (Sigma, St.Louis, Missouri, United States) and the concentration was determined by Bicinchoninic Acid Kit for Protein Determination (Sigma, St.Louis, Missouri, United States). Subsequently, sodium dodecyl sulphate–polyacrylamide gel electrophoresis was performed, and the protein was transferred to polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany), which was further incubated with primary as well as secondary antibodies, and the antibodies applied in Western blot were listed in Table 1 . Then, the bands were visualized by enhanced chemiluminescence substrate: Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific, Massachusetts, United States) and X-ray film (Kodak, New York, United States). And the gray value of Western bolt bands was detected by Image J Software (NIH, Maryland, United States). The SOCS1 protein relative expression was calculated by dividing the gray value of SOCS1 protein by the gray value of β-actin. And the pJAK2 and pSTAT3 protein relative expressions were calculated by dividing the gray value of pJAK2 and pSTAT3 protein by the gray value of JAK2 and STAT3, respectively.
7
Western Blot Analysis of mTOR, Bcl-2, and Bax
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted using RIPA Lysis buffer (Beyotime Institute of Biotechnology, China). The concentration was determined using the Bicinchoninic Acid Kit for Protein Determination (Sigma-Aldrich; Merck KGaA). Samples containing 30 μg protein were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Inc., USA). Primary antibodies against mTOR (cat. No. sc-293089, dilution, 1:1000), p-mTOR (cat. No. sc-293132, dilution, 1:500), Bcl-2 (cat. No. sc-56015, dilution, 1:1000) and Bax (cat. No. sc-6236, dilution, 1:1000) were purchased from Santa Cruz Biotechnology. Cleaved caspase-3 (cat. No. 9661, dilution, 1:1000) was purchased from Cell Signaling Technology, Inc. (USA). After incubation with primary antibodies at room temperature for 2 h, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (cat. No. sc-516102; dilution, 1:10,000; Santa Cruz Biotechnology), following visualization using chemiluminescence (Thermo Fisher Scientific, Inc.). β-actin (cat. No. sc-130301; dilution, 1:2000; Santa Cruz Biotechnology) was used as the control antibody. Signals were analyzed with Quantity One® software, version 4.5 (Bio-Rad Laboratories, Inc.).
8
Western Blot Analysis of RFC3 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein was extracted using NP-40 buffer (Beyotime Institute of Biotechnology, Haimen, China), and the concentration was determined using the Bicinchoninic Acid Kit for Protein Determination (Sigma-Aldrich, St. Louis, MO, USA). Samples containing 50 µg of protein were separated on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The primary antibody anti-RFC3 was purchased from Santa Cruz Biotechnology, Dallas, TX, USA (Cat. no: sc-390293; dilution: 1:1,000). The membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Cat. no: sc-516102; dilution: 1:10,000; Santa Cruz Biotechnology), following visualization with chemiluminescence (Thermo Fisher Scientific, Inc.). GAPDH primary antibody (Cat. no: 2118; dilution: 1:2,000; Cell Signaling Technology, Inc., Danvers, MA, USA) was used as the control antibody.
9
Synthesis and Characterization of Benzoate Esters
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The compounds methyl benzoate (1), n-propyl benzoate(3), n-butyl benzoate (4), methyl 2-naphthoate (7 ), phenylacetate (9 ), ethyl 2-bromobenzoate (10 ), ethyl 3-bromobenzoate (11 ), ethyl 4-bromobenzoate (12 ), methyl 8-fluoro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate (methyl flumazenil ester, 16 ), methyl 4-aminobenzoate (methyl benzocaine ester, 14 ), and methyl [1,1′-biphenyl]-4-carboxylate (17 ) were synthesised at the Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBio®, UFRJ, Brazil) with degree of purity ≥98%. Flumazenil (13 ) was purchased from Cristalia® – Brazil. Formic Acid (96%), acetonitrile and methanol of HPLC grade were purchased from Tedia®-Brazil. Water used for the HPLC analysis was produced using a water purification system (Master System MS2000, Gehaka, Brazil). Ethylbenzoate (2 ), phenylbenzoate (5 ), benzylbenzoate(6 ), methyl 2-phenylacetate (8 ), ethyl 4-aminobenzoate (benzocaine, 13 ), bicinchoninic acid kit for protein determination, ethylenediamine-tetra-acetic-acid (EDTA), monobasic and bibasic potassium phosphate, bis(p-nitrophenyl) phosphate sodium salt, magnesium chloride hexahydrate, β-nicotinamide adenine dinucleotide phosphate (NADP), D-glucose-6-phosphate, glucose-6-phosphate dehydrogenase, tetrahydrofuran (THF) and lithium hydroxide (LiOH) were purchased from Sigma Aldrich (St. Louis, MO, USA).
10
Gelatin Zymography for MMP-2 Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Aneurysm samples were homogenized in protein extraction buffer (0.3 M sucrose; 25 mM Imidazole, 1 mM EDTA, pH 7.2 complete protease inhibitor cocktail 2 and 3, Sigma Aldrich, Søborg, Denmark). Samples were centrifuged for 10 min at 6000× g at 4 °C. Protein concentration was determined by Bicinchoninic Acid Kit for Protein Determination (Sigma Aldrich, Søborg, Denmark) using bovine serum albumin as the standard. A total of 12 µg protein samples and 1.25 µL recombinant MMP-2 (Sigma Aldrich, Søborg, Denmark) were mixed with an equal amount of 2× tris-glycine SDS sample buffer (Thermo Fischer) loaded onto a Novex zymogram gel containing 10% gelatin (Thermo Fisher, Slangerup, Denmark) and proteins were separated by gel electrophoresis at 125 V for 90 min. Proteins were then allowed to refold 30 min in renaturation buffer (Thermo Fisher, Slangerup, Denmark) followed by 24 h at 37 °C in developing buffer (Thermo Fischer, Slangerup, Denmark). Finally, undigested proteins in the gel were stained with simple blue stain (Thermo Fisher) for 30 min. White bands were inverted and quantified in Molecular Imager Image Lab (ChemiDoc WRS+, Biorad, Copenhagen, Denmark).
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!