The total protein of Panc-1 and BxPC-3 cells was extracted by radioimmunoprecipitation assay buffer with protease inhibitor and the concentration of the protein was examined using the Bicinchoninic acid (BCA) Assay kit (Applygen Technologies Inc., Beijing, China). The protein of each sample was then separated by SDS-PAGE (Invitrogen Life Technologies, Carlsbad, CA, USA) and transferred onto the polyvinylidene difluoride membranes (Invitrogen Life Technologies). The membranes were blocked with 5% non-fat milk for 1 h and incubated with the primary antibodies at 4°C overnight. Next, the membranes were probed with the secondary antibodies for 1 h at room temperature. Immunopositive bands were then detected and exposed to film following incubation with an enhanced chemiluminescence system (Applygen Technologies, Inc., Beijing, China). The expression of protein was normalized to the levels of β-actin.
Bca assay kit
Manufactured by Applygen
Sourced in China
The BCA assay kit is a laboratory reagent used to quantify the total protein concentration in a sample. It utilizes the bicinchoninic acid (BCA) method, which is a colorimetric detection technique. The kit provides the necessary reagents and solutions to perform this protein quantification assay.
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Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Imagequant las 500, by GE Healthcare (2 mentions)
Protease inhibitor cocktail, by Selleck Chemicals (2 mentions)
B15001, by Selleck Chemicals (2 mentions)
Ripa lysis buffer, by Solarbio (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions)
Sds page, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence system, by Applygen (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ripa buffer, by Applygen (1 mentions)
Protein a g agarose bead, by Santa Cruz Biotechnology (1 mentions)
Enspire multimode plate reader, by PerkinElmer (1 mentions)
Nitrocellulose membrane, by Applygen (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Trypsin, by Promega (1 mentions)
Ripa lysis buffer, by Applygen (1 mentions)
Phosphatase inhibitor mixture, by Applygen (1 mentions)
A21010, by Abbkine (1 mentions)
A01020, by Abbkine (1 mentions)
14 protocols using bca assay kit
1
Western Blot Analysis of Panc-1 and BxPC-3 Cells
2
Western Blot Analysis of Protein Expression
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After treatment, cells were harvested and lysed with RIPA buffer (C1053, Applygen Technologies, Beijing, China) containing protease/phosphatase inhibitor cocktail (GRF101/102, EpiZyme, Shanghai, China). Protein concentrations were determined using a BCA assay kit (P1511, Applygen Technologies, Beijing, China). Protein samples were separated by SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, United States). After blocked with 5% nonfat milk for 1 h, the membranes were incubated successively with specific primary and secondary antibodies. The protein blots were visualized with “Torchlight” Hypersensitive ECL Western HRP Substrate (17046, Zen Bio, Chengdu, China) and automatic exposure system (Image Quant LAS500, GE, Fairfield, CT, USA).
3
Immunoprecipitation and Western Blotting
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Cells were washed by phosphate buffer saline (PBS) twice and lysed by protein lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40 or Triton X-100, 1 mM EDTA, 10 mM sodium butyrate and protease inhibitors) on ice for 0.5 h. Supernatant was collected after centrifugation and the concentration of protein was measured using Bicinchoninic acid (BCA) assay kit (Applygen, P1511-1). As for immunoprecipitation, protein solution was incubated with corresponding antibodies (3–10 μg) on end-over-end mixer overnight at 4°C, followed by supplementing 30–50 μl protein A/G agarose Beads (Santa Cruz, sc-2001/sc-2002). Subsequently, the reactive systems were incubated on end-over-end mixer for at least 4 h at 4°C, and the beads were washed for 3 times using lysis buffer (without protease inhibitors). Then protein solution or beads were boiled in SDS-PAGE loading buffer with β-mercaptoethanol for 10 min at 100°C and appropriate amount of protein was loaded into SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked in 5% skim milk for at least 1 h and then incubated with indicated antibodies for 4 h at room temperature or overnight at 4°C.
4
Western Blot Protein Analysis
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After the indicated treatments, cells were harvested and resuspended in RIPA buffer for protein extraction. Protein concentration was determined by using a BCA assay kit from APPLYGEN. Aliquots of 80 to100 μg of protein were separated by 10% SDS‐PAGE and then transferred onto PVDF membranes (Merck Millipore Ltd). The membranes were blocked with TBST containing 5% non‐fat milk at room temperature for 1 hours and incubated with the indicated antibodies at 4 °C overnight. Subsequently, the membranes were washed three times with TBST and incubated with secondary antibody conjugated to horseradish peroxidase at room temperature for 1 hour. Finally, the membranes were washed three times with TBST and incubated with ECL reagents. The membranes were examined using a chemiluminescence photodocumentation system photographed and quantitated.
5
P-glycoprotein Function Evaluation
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R123 uptake assay was carried out to identify P-gp function. The confluent ECV304 and bEnd3 cells in 24-well plates were exposed to blank HBSS or HBSS containing P-gp inhibitor verapamil (100 μM) for 30 min at 37°C. Then the medium was removed and P-gp substrate R123 (5 μM) dissolved in HBSS alone or with the inhibitor was added to cells and the incubation lasted for 1 h. After replacing in HBSS with or without the inhibitor, the plate was maintained at 37°C for another 2 h. The cells were lysed with 1% Triton X-100 in PBS for 15min at 37°C and the aliquots were removed for R123 detection using a PerkinElmer EnSpire Multimode Plate Reader (excitation: 485 nM; emission: 535 nM). The protein content was determined by a BCA assay kit (Applygen Technologies Inc., Beijing, China). The R123 concentration was expressed as ng/mg protein.
6
Protein Expression Analysis via Western Blot
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After the indicated treatments, cells were harvested and resuspended in RIPA buffer for protein extraction. Protein concentration was determined using a BCA assay kit from APPLYGEN (Beijing, China). Aliquots of 80–100 μg of protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred onto PVDF membranes (Merck Millipore Ltd., Germany). The membranes were blocked with TBST containing 5% nonfat milk at room temperature for 1 h and incubated with the indicated antibodies at 4 °C overnight. Subsequently, the membranes were washed three times with TBST, and incubated with secondary antibody conjugated to horseradish peroxidase at room temperature for 1 h. Finally, the membranes were washed three times with TBST and incubated with ECL reagents. The membranes were examined using a chemiluminescence photodocumentation system (Tanon, Beijing, China) photographed and quantitated.
7
Western Blot Analysis of Proteins
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Aorta or peritoneal macrophages were isolated from mice and then lysed. The protein content was determined with BCA assay kit (Applygen Technologies, Beijing, China). Equal amounts of proteins were loaded and separated by SDS-PAGE. The proteins transferred on nitrocellulose membranes (Applygen Technologies, Beijing, China) were recognized by primary antibodies and then by secondary antibodies conjugated to horseradish peroxidase. The blots were developed by use of the enhanced chemiluminescence detection reagents. If required, the antibodies bound to membranes were removed by a commercial stripping solution from Applygen Technologies Inc, and then the blots were reprobed with use of other antibodies and developed [24 (link)].
8
Western Blot Analysis of HeLa Cell Lysates
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First, HeLa cells were lysed in RIPA lysis buffer (R0020, Solarbio), which contains 1% protease inhibitor cocktail (B14001; Bimake) and 1% phosphatase inhibitor (B15001; Bimake). The BCA Assay Kit (P1511-1, Applygen) was used to determined the protein concentration of lysate. Proteins were separated on 10% SDS/PAGE gels and then transferred to PVDF membranes (IPVH00010; Millipore). The membranes were blocked for 2 h at room temperature in Tris-buffered saline and 0.1% Tween-20 (TBST) containing 5% skim milk and then were incubated with primary antibodies in the same buffer at 4°C overnight. The protein bands were detected with HRP–conjugated secondary antibodies and Clarity Western ECL Substrate (170-5060; Bio-Rad). The protein bands were visualized with a Bio-Rad System (Bio-Rad, Hercules, CA, U.S.A.). Actin served as a loading control.
9
Western Blot Protocol for RAW264.7 Cells
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After all treatment, RAW264.7 cells were lysed in RIPA lysis buffer (R0020, Solarbio), containing 1% protease inhibitor cocktail (B14001; Bimake) and 1% phosphatase inhibitor (B15001; Bimake). The protein concentration of lysate was determined by using BCA Assay Kit (P1511-1, Applygen). Twenty micrograms of protein was isolated using 10% SDS/PAGE and electro-transferred to PVDF membranes (IPVH00010; Millipore). The membranes were blocked by TBST containing 5% milk for 2 h at room temperature, and probed with various primary antibodies at 4°C overnight. After being washed with TBST at intervals of 10 min, membranes were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. After washing for five times, the immunoreactive bands were visualized using Clarity Western ECL Substrate (170-5060; Bio-Rad). The protein bands were visualized with a Bio-Rad System (Bio-Rad, Hercules, CA, U.S.A.). Actin served as a loading control.
10
Liver Proteome Extraction and Quantification
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The total protein was extracted from liver tissues by using radio immunoprecipitation assay (RIPA) lysis buffer including 10 mM Tris-HCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS). The protein concentration was assessed by bicinchoninic acid (BCA) assay kit (Applygen, Beijing, China) according to the manufacturer's protocol. Two hundred micrograms of protein were divided into two equal parts for each of samples, which were used for quantification of proteome and acetyl-proteome, respectively.
For digestion, the protein was reduced with 5 mM dithiothreitol (Sigma Alrdich, St. Louis, MO, USA) and incubated for 30 min at 56°C, and subsequently alkylated with 55 mM iodoacetamide (Sigma Alrdich) for 15 min at room temperature in darkness. After being washed with 100 mM TEAB buffer (Sigma Alrdich) two times, trypsin (Promega, Beijing, China) was added with a ratio of 1:50 (trypsin:protein) and incubated overnight at room temperature for the first digestion, then added with a ratio of 1:100 (trypsin:protein) and incubated for a further 4 h for the second digestion.
For digestion, the protein was reduced with 5 mM dithiothreitol (Sigma Alrdich, St. Louis, MO, USA) and incubated for 30 min at 56°C, and subsequently alkylated with 55 mM iodoacetamide (Sigma Alrdich) for 15 min at room temperature in darkness. After being washed with 100 mM TEAB buffer (Sigma Alrdich) two times, trypsin (Promega, Beijing, China) was added with a ratio of 1:50 (trypsin:protein) and incubated overnight at room temperature for the first digestion, then added with a ratio of 1:100 (trypsin:protein) and incubated for a further 4 h for the second digestion.
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