The proteins of rat stomach tissues were extracted in ice-cold RIPA lysis buffer (Solarbio, China) by ultrasound, and then determined by the enhanced bicinchoninic acid protein assay kit (Thermo, USA). 30 μg of each sample was loaded on 10% SDS-PAGE gels. And protein blots were transferred onto polyvinylidene fluoride membranes (Milipore, USA). After blocking with 5% non-fat milk, the blots were incubated overnight at 4 °C with a primary antibody: anti-actin (1:5,000, Proteintech Group) and anti-striatin (STRN, 1:1,000, Proteintech Group) antibodies. Then the membranes were washed with a mixture of Tris-buffered saline and Tween 20 (TBST) and incubated at room temperature for 1 h with a secondary antibody conjugated to horseradish peroxidase. Finally, the protein blots were visualized using an enhanced chemiluminescence kit (Millipore, USA).
Enhanced bicinchoninic acid protein assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Enhanced bicinchoninic acid protein assay kit is a colorimetric assay used to quantify total protein concentration in a sample. The kit utilizes a bicinchoninic acid (BCA) reagent to produce a purple-colored reaction that absorbs light at 562 nm, allowing for spectrophotometric measurement of the protein content.
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Lab products found in correlation
Gapdh, by Cell Signaling Technology (3 mentions)
Enhanced chemiluminescence kit, by Merck Group (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Solarbio (2 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (2 mentions)
Anti actin, by Proteintech (1 mentions)
Rock2, by Abcam (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
P0216, by Beyotime (1 mentions)
Rock1, by Abcam (1 mentions)
Secondary antimouse igg antibody, by Beyotime (1 mentions)
Dlc 1, by Abcam (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
β catenin, by Abcam (1 mentions)
Cyclin d1, by Abcam (1 mentions)
Rabbit anti c myc, by Proteintech (1 mentions)
Rabbit anti caspase 9, by Proteintech (1 mentions)
Mouse anti tmem40, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti p53, by Proteintech (1 mentions)
Mouse anti ccnd1, by Proteintech (1 mentions)
6 protocols using enhanced bicinchoninic acid protein assay kit
1
Immunoblotting Analysis of Rat Stomach Proteins
2
Western Blot Analysis of Protein Signaling
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Whole protein lysates of the indicated samples were extracted by RIPA lysis buffer (JRDUN, Shanghai, China) with ethylene diamine tetraacetic acid (EDTA)-free protease inhibitor cocktail (Roche, Mannheim, Germany). The protein concentration was estimated by using an enhanced bicinchoninic acid protein assay kit (Thermo Fisher, Cleveland, OH, USA). Equal amounts of total protein (25 μg) were fractionated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA) overnight. After being blocked with 5% nonfat dry milk (P0216, Beyotime, Shanghai, China) for 1 h at room temperature, the membranes were probed at 4 °C overnight with the primary antibodies (ROCK1 [1:2000; Abcam, Cambridge, UK], ROCK2 [1:1000; Abcam, UK], DLC-1 [1:1000; Abcam, UK], β-catenin [1:5000; Abcam, UK], cyclin D1 [1:10,000; Abcam, UK], and GAPDH [1:2000; Cell Signaling Technology, Beverly, MA, USA]), and then the secondary anti-mouse IgG antibody (1:1000; Beyotime, Shanghai, China) for 1 h at 37 °C. An enhanced chemiluminescence system (Tanon, Guangzhou, China) was used to detect the protein expression value. The protein levels were normalized to GAPDH.
3
Western Blot Analysis of Protein Expression
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The BCa cells and tumor tissue samples lysates were extracted with RIPA cell lysis buffer, and the protein concentration in the lysates was quantified using an enhanced bicinchoninic acid protein assay kit (Thermo Fisher Scientific, MA, USA) with bovine serum albumin as a standard. The total protein extract will be used for western blot analysis. Equal amounts of total protein of tissues or cells were subjected to 10% SDS-PAGE and transferred to a PVDF membrane followed by immunoblotting using the following primary polyclonal antibodies: mouse anti-TMEM40 (Santa Cruz, CA, USA), rabbit anti-p53 (Proteintech Group, INC, USA), rabbit anti-p21 (Proteintech Group, INC, USA), mouse anti-CCND1 (Proteintech Group, INC, USA), rabbit anti-Caspase-3 (Cell Signaling Technology, MA, USA), rabbit anti-Caspase-9 (Proteintech Group, INC, USA), rabbit anti-PARP (Proteintech Group, INC, USA), rabbit anti-c-MYC (Proteintech Group, INC, USA) and GAPDH (Cell Signaling Technology, MA, USA). Specific proteins were detected with enhanced chemiluminescence (ECL, Millipore, USA). Band density was measured (ImageJ software) and normalized to GAPDH.
4
Western Blot Analysis of Cell Signaling
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Cell lysates were extracted with RIPA cell lysis buffer, and the protein concentration in the lysates was quantified using an enhanced bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Protein samples (30–50 μg) were loaded for immunoblotting using antibodies against TMEM140, Bcl2, Bax, intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule1 (VCAM1) and Syndecan1 (Abcam, Cambridge, MA, USA), and GAPDH (Cell Signaling Technology, Danvers, MA, USA). Specific proteins were detected with enhanced chemiluminescence (ECL, Millipore, Bredford, USA). Band density was measured (ImageJ software) and normalized to GAPDH.
5
Western Blot Analysis of Mouse Lung Proteins
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The proteins of mouse lung tissues were extracted in ice-cold RIPA lysis buffer (Solarbio, China) by ultrasound and then determined by the enhanced bicinchoninic acid protein assay kit (Thermo, United States ). Thirty micrograms of each sample was loaded on 10% SDS-PAGE gels, and protein blots were transferred onto polyvinylidene fluoride membranes (Millipore, United States ). After blocking with 5% nonfat milk, the blots were incubated overnight at 4°C with the following primary antibodies: anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Proteintech, 10494-1-AP), anti-intercellular adhesion molecule-1 (ICAM-1, Proteintech, 16174-1-AP) and anti-transferrin (Proteintech, 17435-1-AP). Then, the membranes were washed with a mixture of Tris-buffered saline and Tween 20 (TBST) and incubated at room temperature for 1 h with a secondary antibody conjugated to horseradish peroxidase. Finally, the protein blots were visualized using an enhanced chemiluminescence kit (Millipore, United States ). Three individual samples were analyzed in each group. Bar graphs are relative gray values to GAPDH. The results from each mouse were analyzed by one-way analysis of variance.
6
Intestinal Oxidative Stress Assessment
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Intestinal tissue levels of oxidative stress (OS) were indirectly assessed using MDA content and SOD activity assays. Intestine samples from each group were homogenized in cold saline, with a weight-to-volume ratio of 1:9, and the homogenate was centrifuged at 12,000 x g for 10 min at 4˚C. The protein concentration in the supernatant was determined using the enhanced bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). Measurements of MDA content and SOD activity in tissue homogenates were determined using detection kits (cat. nos. A003-1 and A001-1-1; Nanjing Jiancheng Biological Engineering Institute) according to the manufacturer's protocol. Statistical analyses. Statistical analysis was performed using SPSS 16.0 (SPSS, Inc., Chicago, IL, USA). Data were analyzed by one-way analysis of variance followed by post hoc Tukey's test. All data are representative of at least three independent experiments. Quantitative data are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.
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