The whole hearts were cut into small pieces, then lysed on ice with cell lysis buffer supplemented with protease inhibitor and PMSF for 30 min. Then, samples were centrifuged at 12,000 g at 4 °C for 15 min. The concentration of total protein was determined using BCA protein detection kit (Thermo Scientific, Waltham, MA, USA); 50 μg total protein was separated on 10% SDS-PAGE gel, and then transferred to 0.45 μm PVDF membrane (Amersham Hybond, GE Healthcare, München, Germany). The PVDF membrane was blocked with TBST (Tris buffered saline, 0.2% Tween) buffer and 5% milk at room temperature for 2 h, and then incubated at 4 °C overnight with primary antibodies: rabbit anti-TGF-β1 (1:1,000), rabbit anti-Smad2/3 (1:1,000), rabbit anti-p53 (1:1,000), rabbit anti-Bax (1:1,000), rabbit anti-Bcl-2 (1:1,000), rabbit anti-β-actin (1:5,000). After been washed with TBST buffer, the membrane was incubated with goat anti-rabbit secondary antibody at room temperature for 1 h. Subsequently, the membrane was washed with TBST buffer and tested with ECL Kit (ThermoFisher, USA). Protein expression of β-actin was used as an internal control. Image J software (National Institutes of Health, Bethesda, Maryland, USA) was used for gray-scale quantitative analysis.
Bca protein detection kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
The BCA protein detection kit is a colorimetric assay used to quantify total protein concentration in a sample. The kit utilizes the bicinchoninic acid (BCA) reaction, whereby proteins reduce copper ions (Cu2+) to cuprous ions (Cu+) in an alkaline environment. The resulting purple-colored reaction product is measured spectrophotometrically, and the protein concentration is determined by comparison to a standard curve.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (5 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (5 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Nanodrop 2000 uv spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Gapdh, by Abcam (2 mentions)
Immobilon p pvdf membrane, by Merck Group (2 mentions)
Ecl prime, by GE Healthcare (2 mentions)
Chemidoc mp system, by Bio-Rad (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Stempro osteogenesis differentiation kit, by Thermo Fisher Scientific (2 mentions)
Pnpp alkaline phosphatase assay kit, by BioAssay Systems (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Amersham hybond, by GE Healthcare (1 mentions)
56 protocols using bca protein detection kit
1
Western Blot Analysis of Cardiac Proteins
2
Protein Detection Using BCA Kit
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A BCA Protein Detection Kit (Thermo Scientific™, Shanghai, China) was used to detect the protein concentration. SDS (5×) was added to the total protein, and the mixture was further heated at 100°C for 10 minutes. The protein was isolated by SDS–PAGE and transferred to polyvinylidene fluoride (PVDF) film. Five percent skim milk was sealed at room temperature for 2 hours and further incubated with primary antibody in a shaking bottle at 4°C for 8–12 hours. Then, we washed the film with Tris-buffered saline and Tween 20 (TBST) 3 times, and each wash lasted 10 minutes. After that, the secondary antibody was incubated with film at room temperature for 1 hour and washed with TBST once more 3 times (10 min each time). Proteins were observed by enhanced chemiluminescence.
3
Protein Extraction and Western Blot Analysis
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Radioimmunoprecipitation (RIPA) lysis buffer (Thermo Fisher Scientific, MA, USA) was utilized to extract total cellular protein from CRC cells transfected for 48 h. A bicinchoninic acid (BCA) protein detection kit (Thermo Fisher Scientific, MA, USA) was used to measure the protein content. SDS-PAGE (10%) (Bio-Rad Laboratories, Hercules, CA, USA) was used to separate proteins, after which the proteins were transferred to PVDF membranes. After blocking, the membranes were incubated with primary antibody (1:1000) overnight at 4 °C, followed by incubation with the secondary antibody (1:5000) at room temperature for 2 h before exposure analysis. CDK1 (Abcam, USA) was assessed later, with β-actin as the internal reference (Cell Signalling Technologies, Beverly, MA, USA).
4
Quantifying PGE2 Levels in Nevus Samples
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PGE2 content in plasma samples was determined by ELISA using a PGE2 assay kit (KGE004B, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions as described previously [22 (link)]. For nevus samples, frozen tissue was cut into sub-millimeter pieces and then homogenized in buffer (RD-556) supplied in the assay kit, using a disposable pestle (Kimble 749521-0590) obtained from Sigma-Aldrich, St. Louis, MO) on ice. After homogenization, nevus tissue lysates were sonicated briefly and then microfuged at 10,000 rpm for 10 min at 4 °C. After centrifugation, supernatants were collected, and protein concentrations were determined using a BCA protein detection kit (Thermo Fisher Scientific, Waltham, MA, USA). PGE2 values were normalized to protein content for each sample.
5
Dendritic Cell Maturation and Tumor Antigen Pulsing
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To pulse DC with tumor-associated antigens, tumor cell lysates were prepared through repeated freeze and thaw cycles. After centrifugation, protein concentration was determined in the supernatant using the BCA protein detection kit (Thermo, Bonn, Germany) according to the manufacturer’s instructions. Five days after their generation, DC were pulsed with the different tumor lysates (100 µg/ml).
On day + 6 after their generation, DC were adenovirally transduced with Ad-CD40L at MOI 100. DC were transduced in phosphate-buffered saline (PBS) (PAA) with 2% heat-inactivated autologous serum for 2 h at 37 °C.
On day + 6 after their generation, DC were adenovirally transduced with Ad-CD40L at MOI 100. DC were transduced in phosphate-buffered saline (PBS) (PAA) with 2% heat-inactivated autologous serum for 2 h at 37 °C.
6
Western Blot Analysis of Apoptotic Proteins
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Protein samples were generated by lysing cells with RIPA lysis buffer (Solarbio, Beijing, China). The BCA protein detection kit was utilized to quantify the protein concentrations in the supernatants (Thermo, Waltham, MA, USA). After being separated with 8% SDS, the protein samples were transferred to PVDF membranes. Before overnight incubation at 4 °C with the primary antibody, the membranes were subjected to a 1 h incubation with 5% BSA. The membranes were then exposed to secondary antibodies for 60 min at room temperature, followed by three TBST rinses. The GelView 6000Plus system, produced by Biolight Biotechnology in Guangzhou, China, created the band images. The Western blotting procedure employed several primary antibodies, including NCAPG (24563-1-AP), procured from Proteintech (Wuhan, China). Additionally, p53 (2524), Bax (41162), Bcl-2 (15071), Caspase-9 (9508), and Caspase-3 (9662) were obtained from Cell Signalling Technology (Danvers, MA, USA).
7
Protein Expression Analysis by Western Blot
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The cells were treated with RIPA cleavage buffer containing phosphatase and protease inhibitor (Sigma-Aldrich, USA, #89900). The protein concentration was quantified by the BCA protein detection kit (ThermoFisher, USA, #23225). The protein was separated by 10% SDS-polyacrylamide gel electrophoresis and then transferred to the PVDF membrane. The imprinted membrane was blocked by TBST containing 5% BSA for 1 h. The first antibody against BMP2 (Abcam, UK, #ab214821), P-SMAD1/5/8 (Santa Cruz, USA, #sc-12353P), RUNX2 (Abcam, UK, #ab236639), OCN (Abcam, UK, #ab133612), PPARγ(Abcam, UK, #ab178860), and β-actin (Abcam, UK, #ab8226) was incubated at 4°C for 14 h, and then the membrane was washed and incubated with goat anti-rabbit (ThermoFisher, USA, #A21076) or goat anti-mouse (ThermoFisher, USA, #A21094) secondary antibody at room temperature for 1 h. The protein was observed by the ECL Western blotting imaging system (ThermoFisher, USA, #32209). The Image Lab software (Bio-Rad, USA) was used to quantify the gray value of protein bands.
8
Protein Enrichment and Preparation
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The samples were concentrated using a speed vac to minimize the water content, and the proteins were precipitated using ice-cold ethanol (Decon Labs, PA 19406). The protein pellet was dissolved in urea buffer (6 M urea in 0.1 M Tris/HCl, pH 8.5) and the concentration of the protein mixture was calculated using BCA protein detection kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the kit protocol using urea buffer as a blank. The protein mixture was denaturated by adding 1 M Dithiothretiol, (DTT, Acros Organics, Morris Plains, NJ, USA) in 100:1 v/v ratio to the reaction mixture and heated at 95 °C for five minutes then cooled and added 1 M iodoacetamide (IAM, from Acros Organics, Morris Plains, NJ, USA) in the ratio of 50:1, v/v at 37 °C for 1 h (reductive alkylation reagents were purchased from Acros Organics, Morris Plains, NJ, USA). The reduced and alkylated samples were desalted, and buffer exchanged with 50 mM ammonium bicarbonate, pH 8.0 buffer, using Microcon-10 kDa (YM-10, 0.5 mL, Millipore, Burlington, MA, USA) and aliquoted to three parts.
All other reagents used here, which are not categorically mentioned were additionally purchased from Sigma-Aldrich (St. Louis, MO, USA).
All other reagents used here, which are not categorically mentioned were additionally purchased from Sigma-Aldrich (St. Louis, MO, USA).
9
Western Blot Analysis of Brain Tissue Proteins
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Mouse brain tissues were homogenized in ice-cold RIPA lysis buffer (1 × PBS buffer containing 1% NP-40, 0.5% Na deoxycholate, and 0.1% SDS) with a protease & phosphatase inhibitor cocktail (Thermo Fisher, cat#: UG280144) [47 (link)]. Protein concentrations were determined using the BCA protein detection kit (Thermo Fisher, cat#: 23227), and equal amounts of protein (10 µg per lane for tissue lysates) were resolved on denaturing 10% SDS–PAGE gels and transferred by electroblotting to PVDF membranes (Millipore, cat#: IPVH00010, Burlington, MA). Membranes were incubated with anti-VEGFR1 (Abcam, cat#: ab2350, 1:500), anti-VEGFR2 (Abcam, cat#: ab221679, 1:1000), anti-Cgn (Sigma, cat#: HPA027657, 1:500, St. Louis, MO), anti-ZO-1 (Invitrogen, cat#: 61–7300, 1:1000, Carlsbad, CA), anti-Claudin 5 (Invitrogen, cat#: 34–1600, 1:1000), or anti-Actin (Millipore, cat#: MAB1501R, 1:5000). The membranes were washed with PBST (0.2% Tween-20 in PBS), incubated with a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Earthox, cat#: E030110-01, 1:20,000, Millbrae, CA) or HRP goat anti-rabbit IgG (Earthox, cat#: E030120-01, 1:20,000) for 1 h, washed again, and incubated with ECL detection reagents (Millipore, cat#: WBKLS0500). Densitometry analysis was performed using the ImageJ (version 1.52p, NIH) software.
10
Protein Expression Analysis of Sciatic Nerve
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The total protein of sciatic nerve tissue was extracted. First, the nucleus protein was extracted by nucleus protein extraction kit (Beyotime Bio, China). The content was detected by BCA protein detection kit (Thermo, Waltham, MA) according to the instructions. Then, the protein (30 µg/samples) with 10% SDS-PAGE was isolated and transferred to the PVDF membrane, sealed in 5% skim milk under 25°C for 1 h, and incubated with primary NF-B P65 (1:2000, Abcam Biotech, Cambridge, MA, USA), Caspase1 (1:1000, Abcam), Pro-Caspase1 (1:1000, Cusabiao, Wuhan, China), GSDMD-N (1:1000, Abcam), Klotho (1:1000, Abcam), NLRP3 (1:1000, Abcam), GAPDH (1:2500, Abcam) and H3 (1:2000, Abcam). And then, it was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Beyotime, Shanghai, China) for 1 h. After that, protein bands were detected with a ECL detection kit (Beyotime Biothech, Shanghai, China), GAPDH or H3 was taken as control.
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