Whole colostrum samples (n = 1 sample/treatment) were thawed at 4°C overnight and fractionated and enriched as described by Tacoma et al. (2016) (link). Briefly, mammalian protease inhibitor (Protease Inhibitor Cocktail, Sigma) was added to samples before centrifugation at 4,000 × g for 15 min at 4°C. The skim fraction was isolated and underwent a second centrifugation using the same conditions. The separated skim milk fraction was collected and to this fraction 60 mM of CaCl2 was added for CN depletion as outlined by Kunz and Lonnerdal (1990) . The pH of each sample was adjusted to using 30% acetic acid (Fisher Scientific, Fair Lawn, NJ) and centrifuged at 189,000 × g for 70 min at 4°C. The supernatant was collected, frozen to −80°C, then lyophilized. One hundred milligrams of the resulting whey powder was reconstituted in PBS, and the protein concentration of each sample was determined using the bicinchoninic acid assay kit (Pierce Biotechnology, Rockford, IL). The low abundance protein fraction of each sample was enriched using the ProteoMiner kit (Bio-Rad Laboratories, Hercules, CA), where 10 mg of protein was loaded onto each column and enrichment was performed as per kit instructions. The protein concentration of each sample was determined using the bicinchoninic acid assay kit (Pierce Biotechnology).
Bicinchoninic acid assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Germany
The Bicinchoninic acid (BCA) assay kit is a colorimetric method for determining the total protein concentration in a sample. The kit uses a proprietary reagent containing bicinchoninic acid, which forms a purple-colored complex with proteins in an alkaline environment. The intensity of the color is proportional to the protein concentration and can be measured using a spectrophotometer.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (15 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (13 mentions)
Protease inhibitor cocktail, by Merck Group (11 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (7 mentions)
Ripa lysis buffer, by Beyotime (6 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (6 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Polyvinylidene difluoride membrane, by Merck Group (6 mentions)
Ab205718, by Abcam (5 mentions)
Radioimmunoprecipitation assay buffer, by Merck Group (5 mentions)
β actin, by Santa Cruz Biotechnology (5 mentions)
Protease inhibitor, by Roche (5 mentions)
Ezripa buffer kit, by ATTO Corporation (4 mentions)
β actin, by Cell Signaling Technology (4 mentions)
Odyssey infrared imaging system, by LI COR (4 mentions)
Ripa buffer, by Beyotime (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Nitrocellulose membrane, by Merck Group (4 mentions)
Anti β actin, by Abcam (3 mentions)
Pvdf membrane, by Thermo Fisher Scientific (3 mentions)
250 protocols using bicinchoninic acid assay kit
1
Enrichment of Colostrum Whey Proteins
2
Enrichment of Colostrum Whey Proteins
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Whole colostrum samples (n = 1 sample/treatment) were thawed at 4°C overnight and fractionated and enriched as described by Tacoma et al. (2016) (link). Briefly, mammalian protease inhibitor (Protease Inhibitor Cocktail, Sigma) was added to samples before centrifugation at 4,000 × g for 15 min at 4°C. The skim fraction was isolated and underwent a second centrifugation using the same conditions. The separated skim milk fraction was collected and to this fraction 60 mM of CaCl2 was added for CN depletion as outlined by Kunz and Lonnerdal (1990) . The pH of each sample was adjusted to using 30% acetic acid (Fisher Scientific, Fair Lawn, NJ) and centrifuged at 189,000 × g for 70 min at 4°C. The supernatant was collected, frozen to −80°C, then lyophilized. One hundred milligrams of the resulting whey powder was reconstituted in PBS, and the protein concentration of each sample was determined using the bicinchoninic acid assay kit (Pierce Biotechnology, Rockford, IL). The low abundance protein fraction of each sample was enriched using the ProteoMiner kit (Bio-Rad Laboratories, Hercules, CA), where 10 mg of protein was loaded onto each column and enrichment was performed as per kit instructions. The protein concentration of each sample was determined using the bicinchoninic acid assay kit (Pierce Biotechnology).
3
Protein Expression and Western Blot Analysis
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Cells were lysed in RIPA Lysis buffer (Invitrogen). Total proteins were quantified using a bicinchoninic acid assay kit (Invitrogen), separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidenefluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% skim milk in Tris-buffered saline Tween (TBST) for 2 h, and incubated with specific primary antibodies, including anti-ERβ, -NF-κB p65, -p-NF-κB p65, -vascular endothelial growth factor A (VEGF-A), -matrix metalloproteinase 2 (MMP-2), and -GAPDH (Rabbit anti-human, 1:1000, Abcam, Cambridge, MA, USA) overnight at 4°C. Then the membrane was washed with TBST for three times, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-rabbit, 1:5000, Abcam) for 1 h at 25°C. The protein bands were visualized using HRP color development kit (Invitrogen).
4
Protein Quantification and Cytokine Analysis
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We collected the serum and cell culture medium supernatant. We used a bicinchoninic acid assay kit (Invitrogen, Carlsbad, CA, United States) to determine protein concentration. We conducted ELISA analysis using the TNF-α, IL-1β, and IL-18 ELISA Kit (Anoric-Bio, Tianjin, China) according to the manufacturer’s instructions.
5
Quantitative Protein Analysis of MCF-7 Cells
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MCF-7 breast cancer cells were homogenized and lysed in RIPA buffer containing protease and phosphatase inhibitors (cOmplete™ Protease Inhibitor Cocktail, Roche, Shanghai, China). We measured the protein concentration of the homogenized sample using a bicinchoninic acid assay kit (Invitrogen), according to the manufacturer’s recommendations. The samples were boiled for 10min in a water bath. We separated the protein in the extracts using 10%SDS-PAGE and transferred them to nitrocellulose membranes. Subsequently, membranes were blocked with 3% BSA, washed in 1×TBST and incubated overnight with primary antibodies. The primary antibodies are listed in Supplemental materials
6
Western Blot Analysis of Protein Extracts
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For western blot analysis, the total protein extracts from cells (RIPA, Beyotime Biotechnology, Shanghai, China) and tissue (One Step Animal Tissue Active Protein Extraction Kit, Sangon Biotech) were quantified by a bicinchoninic acid assay kit (Invitrogen). After that, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride membranes. Then, the membranes were blocked with Tris-buffered saline supplementing with 0.1% Tween 20 (TBST) and 5% fat-free milk for 1 h and incubated with corresponding antibodies at 4°C overnight. The membranes were incubated with secondary antibodies at room temperature after washing with TBST 3 times. The membranes were measured by an image analysis system (Image-Pro Plus 6.0, Media Cybernetics, Rockville, MD, USA) after incubating with a high-signal electrochemiluminescence kit (Fdbio Science, Hangzhou, China).
7
Protein Extraction and Western Blot Analysis
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Total protein was extracted using a urea buffer containing a protease inhibitor cocktail (Bio-Rad Laboratories, Inc.). Total protein in each sample was measured using a bicinchoninic acid assay kit (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Equal amounts of protein (40 µg) were resolved using 10% SDS-PAGE gel electrophoresis and electro-transferred onto a PVDF membrane. Subsequently, the membrane was blocked for 60 min at room temperature with TBST containing 5% skim milk. The membrane was incubated overnight at 4°C with the following primary antibodies: Anti-TLR2 (cat. no. ab16894; 1:1,000; Abcam), anti-IL-16 (cat. no. ab207181; 1:1,000; Abcam) and anti-β-actin (cat. no. ab115777; 1:1,000; Abcam). After washing with PBS, the membrane was incubated with horseradish peroxide-labeled secondary antibodies (cat. no. 7076s; 1:10,000; Cell Signaling Technology Inc.) for 1 h at 37°C and was subsequently developed using an ECL method with the ECL™ Western Blotting Detection Reagents (Sigma-Aldrich; Merck KGaA). The membrane was visualized using a Gel Doc EZ Imager (Bio-Rad Laboratories, Inc.) and the protein bands were analyzed using ImageJ software (version 1.8.0; National Institutes of Health) using β-actin as the loading control.
8
Protein Expression Analysis by Western Blot
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Protein levels were detected by western blotting referring to the protocols in a previous report [20 (link)]. In brief, HaCaT cells were lysed in radio-immunoprecipitation assay buffer (Beyotime, Shanghai, China), and extracted protein was quantified by bicinchoninic acid assay kit (Thermo Fisher Scientific). Protein samples (20 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transfer of polyvinylidene fluoride membranes (Thermo Fisher Scientific). After incubating in 5% fat-free milk, the membranes were incubated with primary antibodies for IGF2BP2 (ab129071, 1:3000 dilution, Abcam, Cambridge, UK), HPSE (ab254254, 1:3000 dilution, Abcam), or β-actin (ab8227, 1:3000 dilution, Abcam) overnight, and then incubated with horseradish peroxidase-conjugated IgG (ab205718, 1:8000 dilution, Abcam) for 2 h, followed by exposure to enhanced chemiluminescence kit (Beyotime). The visualized blots were analyzed via Image J software (NIH, Bethesda, MD, USA), with β-actin as a reference.
9
Western Blot Analysis of Bcl-2 and Bax
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Total protein was extracted from cells using the radioimmunoprecipitation assay buffer kit (Thermo Fisher Scientific, Inc.). Protein concentration was quantified using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc.) prior to separation via SDS-PAGE on a 10% gel at 30 µg protein/lane. The separated proteins were then transferred onto polyvinylidene fluoride membranes and blocked with 5% non-fat milk at room temperature for 1 h. Following blocking, the membranes were then incubated with primary antibodies against Bcl-2 (cat no. 4223), Bax (cat no. 5023) and β-actin (cat no. 4970; all dilution: 1:1,000; Cell Signaling Technology, Inc.) at 4°C overnight. The membranes were washed with phosphate buffer saline (PBS)-0.05% Tween 20 5 times and then incubated with a horseradish peroxidase-conjugated anti-rabbit immunoglobulin G secondary antibody (cat no. 7074; dilution: 1:2,000; Cell Signaling Technology, Inc.) at room temperature for 2 h. The protein bands were visualized using an enhanced chemiluminescence kit (Applygen Technologies, Inc.) according to manufacturer's protocols. Densitometry was performed using the ImageJ software (version 1.38X; National Institutes of Health).
10
Protein Expression Analysis in Osteoarthritis
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Total protein was extracted from OA tissues and CHON-001 cells using RIPA lysis buffer (Beyotime Institute of Biotechnology) and quantified using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc.). Proteins (40 µg/lane) were separated by SDS-PAGE on 10% gels and were transferred onto PVDF membranes (Thermo Fisher Scientific, Inc.). Subsequently, the membranes were blocked with 5% skim milk in TBS-Tween (10%) for 1 h at room temperature. The membranes were then incubated overnight at 4°C with primary antibodies targeted against: Collagen II (1:1,000; cat. no. ab325034; Abcam), aggrecan (1:1,000; cat. no. ab1816028; Abcam), MMP13 (1:1,000; cat. no. ab25367; Abcam), p21 (1:1,000; cat. no. ab14323; Abcam), cleaved caspase-3 (1:1,000; cat. no. ab43583; Abcam) and β-actin (1:1,000; cat. no. ab06789; Abcam). Following primary antibody incubation, the membranes were incubated with a HRP-conjugated secondary antibody (1:5,000; cat. no. ab20876; Abcam) for 1 h at room temperature. Protein bands were detected using an ECL kit (Thermo Fisher Scientific, Inc.). Protein expression was semi-quantified using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc.) with β-actin as the internal control.
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