Vascular smooth muscle cells were lysed in RIPA lysis buffer and incubated for 30 min on ice. The supernatant was collected after centrifugation. VSMCs in the media layer of mice were extracted as follows: mice arteries were opened longitudinally, and the endothelium and vascular adventitia were scraped off, then lysed with lysis buffer. Protein concentrations were tested by bicinchoninic acid assays (Beyotime, Shanghai, China). Proteins were separated by SDS-polyacrylamide gels and transferred onto PVDF membranes. The blots were blocked with 5% nonfat milk in 0.05% tween 20 in PBS and incubated with primary antibodies overnight at 4°C. Following incubation overnight, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies and detected by an enhanced chemiluminescence detection kit.
Bicinchoninic acid assay
Manufactured by Beyotime
Sourced in China
The Bicinchoninic Acid (BCA) Assay is a colorimetric detection and quantification method used to measure the total protein concentration in a sample. It relies on the reduction of Cu2+ to Cu+ by proteins in an alkaline medium, and the subsequent chelation of the Cu+ ions with bicinchoninic acid to produce a purple-colored complex that absorbs light at 562 nm.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (18 mentions)
Pvdf membrane, by Merck Group (18 mentions)
Polyvinylidene fluoride membrane, by Merck Group (17 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (17 mentions)
Ripa buffer, by Beyotime (16 mentions)
β actin, by Santa Cruz Biotechnology (9 mentions)
Nitrocellulose membrane, by Merck Group (8 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (7 mentions)
Polyvinylidene difluoride membrane, by Merck Group (7 mentions)
Image pro plus software, by Media Cybernetics (6 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (6 mentions)
Ripa buffer, by Thermo Fisher Scientific (6 mentions)
Ecl plus, by Thermo Fisher Scientific (6 mentions)
Bcl 2, by Cell Signaling Technology (5 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (4 mentions)
Protease inhibitor cocktail, by Beyotime (4 mentions)
Quantity one software, by Bio-Rad (4 mentions)
Ab6721, by Abcam (4 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (4 mentions)
Gapdh, by Abcam (4 mentions)
206 protocols using bicinchoninic acid assay
1
Western Blot Analysis of Vascular Smooth Muscle Cells
2
NRSF Quantification in Hippocampal Tissue
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NRSF was measured after SOD and MDA assays. Total proteins in the collected hippocampal tissues were isolated using cell lysis solution (Beyotime). Protein concentrations were determined by Bicinchoninic Acid assays (Beyotime). Equal amounts of protein were separated on 10% sodium dodecyl sulfonate-polyacrylamide gels and transferred to polyvinylidene fluoride membranes. The membranes were blocked in Tris buffered saline containing nonfat milk (10%) and 0.1% Tween-20 for 1 hour. The following primary antibodies were used: polyclonal rabbit anti-NRSF (Bioss) diluted 1:500, and mouse anti-β-actin (Beyotime) diluted 1:1000. After incubation with primary antibodies for 24 hours at 4°, the membranes were washed three times with Tris buffered saline containing 0.1% Tween-20 for 5 minutes and then incubated with goat anti-rabbit and goat anti-mouse IgG horseradish peroxidase antibody (Beyotime) at a dilution of 1:1000 for 1 hour at room temperature. The chemiluminescence signals were captured using a LAS 4000 mini (General Electric Company, Japan), and the bands were quantified by densitometry using the ImageJ system (National Institutes of Health, Maryland, USA). Grayscale values were measured and normalized to β-actin in the same sample.
3
Cellular Cholesterol Quantification
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Total cellular cholesterol was measured with the Cholesterol Quantitation Kit from Applygen (Peking, China) according to the manufacturer's protocol. The activities of cholesterol esterase and cholesterol oxidase, and the amount of H2O2 produced were quantified using this colorimetric assay. The values were normalized against the total protein concentration in each sample, which was estimated using Bicinchoninic Acid assays (Beyotime Biotechnology, China).
4
Protein Extraction and Western Blot Analysis
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Total protein was extracted using RIPA buffer (Beyotime, Shanghai, China) supplemented with Protease Inhibitor Cocktail and PhosSTOP (Beyotime, Shanghai, China). The total cell protein concentrations were measured with bicinchoninic acid assays (Beyotime, Shanghai, China) with bovine serum albumin (BSA) as a standard. The lysates were separated in a 10% sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) gel, transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA), and incubated with antibodies against RAB43, Snail, N-cadherin, E-cadherin, vimentin, GAPDH, Bax, Bcl-2, PI3K, AKT and P-AKT (all from Cell Signaling Technology, MA, USA) diluted at 1:1000.
5
Western Blot Analysis of Autophagy Markers
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Radioimmunoprecipitation assay buffer (RIPA) was applied to lyse cells. Total proteins were then harvested and quantified with bicinchoninic acid assays (Beyotime, Shanghai, China). The target proteins were separated through 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Merck Millipore, Burlington, MA, USA). The membranes were blocked with nonfat milk, incubated with primary antibodies, and then incubated with secondary antibody diluted at a ratio of 1:10,000 (Jackson ImmunoResearch, West Grove, PA, USA). The primary antibodies were anti-LC3B (2775, Cell Signaling Technology, Beverly, MA, USA), anti-p62 (88588, Cell Signaling Technology), anti-HuR (ab28660, Abcam, Cambridge, MA, USA), anti-SFRS1 (ab133689), anti-FMRP (ab17722), anti-ALKBH5 (ab195377), anti-IGF2BP1 (ab82968), anti-LIN28A (ab46020), anti-FTH1 (ab65080), anti-NCOA4 (ab86707), anti-BCL-2 (ab32124), anti-BECN1 (ab62557), and anti-GAPDH (ab9485). The protein signals were visualized with enhanced chemiluminescence detection reagents (ECL, Millipore, Burlington, MA, USA) and quantified with Image Lab software (Bio-Rad, Hercules, CA, USA).
6
Protein Extraction and Western Blot Analysis
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The total proteins of ESCs were collected by radioimmunoprecipitation assay buffer (RIPA) (Beyotime Biotechnology, China) and quantified by bicinchoninic acid assays (Beyotime, Shanghai, China). The protein samples were denatured after incubation at 95°C for 10 minutes. Target proteins were separated by 12% SDS-PAGE and transferred to a polyvinylidene fluoride membrane, sealed with 5% skim milk at room temperature for 1 hour, incubated overnight with the primary antibody, and then soaked with the secondary antibody. The primary antibody used included the following: anti-HIF-1α (Affinity, AF1009, 1 : 1000), anti-GLI1 (Abcam, ab217326, 1 : 1000), anti-MMP2 (Affinity, AF5330, 1 : 1000), anti-MMP9 (Affinity, AF5228, 1 : 1000), anti-GAPDH (ProteinTech, 10494-1-AP, 1 : 20000). The commercial sources and characteristics of antibodies used are summarized in Supplemental Table 3 .
7
Western Blot Analysis of Tight Junction Proteins
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Cells were lysed using Radio Immunoprecipitation Assay lysis buffer (Beyotime), supplemented with protease inhibitor cocktail (Beyotime) and phenylmethylsulfonyl uoride. The protein concentration was quanti ed using bicinchoninic acid assays (Beyotime). Next, 20-40 µg protein from cell lysates was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Beyotime) and transferred to polyvinylidene uoride membranes (Millipore, MA, USA). The membranes were incubated with primary antibodies at 4 °C overnight and then with secondary antibodies for 1 h. Finally, the target blots were visualized using ECL chemiluminescent reagent (Meilunbio, China), and data were analyzed with Image Lab Software. The primary antibodies used were as follows: anti-ZO
8
Western Blot Analysis of CRC Proteins
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Proteins were isolated from CRC cells and tissues using RIPA lysis buffer (Thermo Scientific, USA) for Western blot, following the manufacturer's instructions. Protein concentrations were determined using the bicinchoninic acid assay (Beyotime Biotechnology, China). Equal amounts of protein were separated using SDS-PAGE on a 10% gel, then transferred onto polyvinylidene difluoride membranes, followed by blocking with 5% skim milk for 2 h. After incubation with primary antibodies (including MNX1 antibody: 1:1000, Abcam, ab92606, USA; E2F4 antibody: 1:500, Proteintech, 10923-1-AP, China; β-actin: 1:2000, Proteintech, 66009-1-Ig, China), the samples were washed 4 times (10 min for each wash), then, the membranes were incubated with secondary antibodies (Millipore, Bedford, USA) for 2 h at room temperature. We wrapped the PVDF membrane in the film. Under the condition of red light in the darkroom, the film is processed by developer and fixative. Once dried, the protein bands can be seen directly. Then the film image is converted into a grayscale image by Photoshop software. In the process, the background of the image is converted to gray from blue.
9
Western Blot Analysis of PRMI8226 and U266 Cells
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Following exposure to CM (0, 0.5, 1, 2 µmol/l for PRMI8226 cells, and 0, 2, 4, 8 µmol/l for U266 cells) for 48 h, the cells were harvested and washed with ice-cold PBS. Proteins were extracted using ice-cold radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) with phenylmethylsulfonyl fluoride. Following a brief sonication, the cell lysates were kept on ice and subsequently centrifuged at 15,984 × g for 10 min. Supernatant was collected and the protein concentrations were quantified with a bicinchoninic acid assay (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Total protein (40 µg/lane) was separated using 10–12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membrane was subsequently blocked with TBST (0.05% Tween-20) containing 5% skimmed milk for 1 h and probed overnight with the corresponding primary antibodies (diluted in primary antibody dilution buffer from Beyotime Institute of Biotechnology) at 4°C. Subsequently the membrane was washed with TBST, exposed to the corresponding HRP-conjugated secondary antibodies for 1 h at room temperature, and detected by enhanced chemiluminescence (EMD Millipore).
10
Western Blot Analysis of CENPF and α-Tubulin
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Cells were lysed using RIPA buffer (50 mM Tris–HCl [PH7.4], 1% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, and phosphatase and protease inhibitors) and the concentration of protein was assessed using a bicinchoninic acid assay (Beyotime Institute of Biotechnology). Lysates were sonicated, centrifuged, and subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Proteins were transferred onto a nitrocellulose membrane (Osmonics Inc., USA). Membranes were subsequently probed with primary anti-CENPF (1:1000; cat. no. ab5; Abcam) or anti-α-Tubulin (1:500; cat. no. sc-5286; Santa Cruz) by overnight incubation at 4 °C. Imaging of immunoblots were performed with a LICOR system using respective fluorescence antibody: IRDye® 800CW Donkey anti-Mouse IgG Secondary Antibody (1:15,000; cat. no. C50422-04; LICOR).
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