Proteins samples isolated from the phenol-ethanol supernatant obtained after DNA precipitation during RNA isolation procedure were quantified using BCA (Bicinchoninic acid, Sigma-Aldrich, USA) assay. Protein homogenates were subjected to 12% SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions followed by electro-transfer on to PVDF membrane (Millipore Inc., USA) using a wet transfer system (Bio-Rad Laboratories Inc., USA). The membranes were then blocked with 5% skim milk, washed and probed with anti- human DPT antibody (1:1000 dilutions; sc-376863) for 1 hour at 37°C. Appropriate secondary antibody conjugated with alkaline phosphatase (sc-2037) was added to the membrane and incubated for 1 hour at 37°C. All antibodies were purchased from Santa Cruz Biotechnology Inc. (USA). Bands were visualized using BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) solution (Sigma-Aldrich, USA) and imaged with GelDoc XR documentation system (Bio-Rad Laboratories Inc, USA).
Bicinchoninic acid (bca)
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Italy, India
The BCA (Bicinchoninic Acid) is a laboratory reagent used for the quantitative colorimetric determination of protein concentration. It functions by forming a purple-colored complex with copper ions in an alkaline environment, where the intensity of the color is proportional to the protein concentration in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (11 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (11 mentions)
Triton x 100, by Merck Group (8 mentions)
Glycerol, by Merck Group (7 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Dithiothreitol (dtt), by Merck Group (5 mentions)
Iodoacetamide, by Merck Group (5 mentions)
Penicillin, by Merck Group (5 mentions)
Chloroform, by Merck Group (5 mentions)
Ripa buffer, by Merck Group (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Hepes, by Merck Group (4 mentions)
Copper sulfate, by Merck Group (4 mentions)
Sulfuric acid, by Merck Group (4 mentions)
Milli q system, by Merck Group (4 mentions)
Ethanol, by Merck Group (4 mentions)
Streptomycin, by Merck Group (3 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Trichloroacetic acid, by Merck Group (3 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (3 mentions)
157 protocols using bicinchoninic acid (bca)
1
Quantification and Analysis of DPT Protein
2
Atherosclerosis Inhibition by BCA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Forty male 8-week-old apoE−/− mice on a C57BL/6 background were purchased from Changzhou Cavens Lab Animal Co., Ltd (Jiangsu, China) and housed under a 12 h light/dark cycle in an environmentally controlled room (24 ± 2°C, 60% humidity). Mice were fed a Western diet containing 21% fat and 0.3% cholesterol and randomized into two groups (n = 20 per group): control group and BCA group. Mice in BCA group were intragastrically administered with 50 mg/kg BCA (Sigma-Aldrich, St. Louis, MO, USA) dissolved in 1% dimethyl sulfoxide (DMSO) twice every week, and control mice were treated with an equal volume of vehicle. Body weight of each mouse was recorded once every two weeks. Twelve weeks later, all mice were sacrificed, and the heart, aorta, and blood were collected. Sodium pentobarbital anesthesia was conducted throughout all surgeries. Prior to euthanasia, 4% thioglycollate broth was intraperitoneally injected into mice, followed by injection with 5 mL of sterile PBS for the collection of mouse peritoneal macrophages (MPMs). After centrifugation (300 rpm, 5 min, 4°C), the isolated MPMs were counted and then cultured in RPMI 1640 medium (Gibco, Grand Island, NE, USA) containing 10% fetal bovine serum (FBS, Gibco). All experiments were conducted according to protocols approved by the Animal Care and Use Committee at the Second Affiliated Hospital of Hainan Medical University.
3
Quantifying Protein Levels by BCA Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein quantity was measured by Bicinchoninic acid (BCA) (Sigma) assay. Briefly, 2% (v/v) copper sulphate (Sigma) was added to BCA (sigma) solution. A 200 μl of this solution was added to 10 μl of protein sample and incubated at 37 °C in the dark for 30 min. The absorbance was read at 570 nm using a Benchmark 96 well plate reader.
4
Protein Extraction and Digestion Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rapigest™ SF was purchased from Waters (Milford, MA, USA). Neat formic acid (FA), dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate (ABC), and bicinchoninic acid (BCA) solution were obtained from Sigma-Aldrich (St. Louis, MO, USA). Sequencing-grade modified trypsin was acquired from Promega (Madison, WI, USA). High-performance liquid chromatography (HPLC)-grade water, 0.1% FA in HPLC water, 0.1% FA in acetonitrile (ACN), and methanol were purchased from Thermo Fisher Scientific (Bremen, Germany).
5
Culturing HT-29 Colon Cancer Cells and Astrocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The HT-29 human colon cancer cell line were acquired from the American Type Culture Collection. HT-29 were cultured in Complete Medium (CM) composed by high-glucose DMEM with 10% FBS, 2 mM L-glutamine, 100 IU/mL penicillin, and 100 μg mL−1 streptomycin (Sigma-Aldrich, Italy). Primary cultures of astrocytes were obtained according to the method described by51 (link). Full description is reported in the supplementary material. Astrocytes were starved in serum-free DMEM overnight before all treatments. Bicinchoninic acid (Sigma-Aldrich, Italy) assay was used to measure protein homogenate concentrations.
6
Quantitative Proteomic Analysis with ICAT
7
Western Blot Analysis of Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were grown to log-phase, washed twice with ice-cold PBS, and lysed in SDS lysis buffer according to the manufacturer's protocol (Cell Signaling Technology, Danvers, MA, USA). Protein concentration was quantified using the BCA protein assay reagent bicinchoninic acid (Sigma). Cell lysates containing 25 μg of protein were separated by SDS-PAGE and transferred to PVDF membranes (Millipore). Membranes were immunoblotted with antibodies against phospho-Akt (S473), total Akt, phospho-p70S6K (T389), total p70S6K, phospho-rpS6 (S235/236), total rpS6, phospho-ERK (T202/Y204), total ERK, (all from Cell Signaling Technology), tubulin (Sigma), CK5 (Leica), and actin (Millipore), using SuperSignal West Pico (Thermo Scientific, Waltham, MA, USA) or ECL advance (GE Healthcare, Auckland, New Zealand) chemiluminescence reagents. Antibody reactivity was visualized using the chemiluminescence detection system by Fujifilm Las-3000.
8
Anandamide and 2-AG Signaling in HOBs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HOBs were cultured for 24 hours and then treated with vehicle (0.1% ethanol), 10μM anandamide or 10μM 2-AG for 20 minutes. The cells were lysed in RIPA buffer (Roche Applied Science, UK), and the supernatant was used for signalling protein analysis. Levels of phosphorylated signalling proteins (ERK/MAP kinase 1/2, Akt, STAT3, JNK, p70 S6 kinase, NFκB, STAT5 A/B, CREB and p38) were measured using a Milliplex map multi-pathway magnetic bead 9-plex signalling multiplex assay kit (48-680MAG, Millipore, USA). The plate was read using a MAGPIX analyser (Luminex, USA) and sample values were interpolated from known standards using the built-in xPONENT software. All results were normalised to the protein content from the corresponding sample as measured using a bicinchoninic acid protein assay. A 1:50 ratio of copper (II) sulphate pentahydrate 4% solution: bicinchoninic acid (Sigma, UK) was added to each sample and incubated at 37°C on a shaker. Samples were measured in duplicate at 562nm and interpolated from a standard curve of bovine serum albumin (Sigma, UK).
9
Optimized Lipid Extraction Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell culture plastic material was purchased from TPP (Trasadingen, Switzerland), and fetal bovine serum (FBS) and trypsin-EDTA were acquired from Biowest (Riverside, MO, USA). LC-MS grade methanol was purchased from Honeywell Burdick and Jackson (Morristown, NJ, USA); 2-propanol and sodium hydroxide were purchased from Merck (Toluca, Mexico); chloroform, HEPES, urea, bicinchoninic acid, and sodium tartrate dihydrate were purchased from Sigma-Aldrich (Toluca, Mexico); sodium carbonate and sodium bicarbonate were purchased from Fermont (Mexico City, Mexico); and cupric sulfate pentahydrate was purchased from Golden Bell (Mexico City, Mexico). Ultrapure water was obtained from a Direct-Q 3 system (Millipore, Burlington, MA, USA). HO-AAVPA was synthesized as published by Prestegui-Martel et al. (2016) with a few modifications [37 (link)]. VPA, formic acid, ammonium acetate, and ammonium formate were acquired from Sigma-Aldrich (Toluca, México).
10
Western Blot Protein Analysis Protocol
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!