Liver tissue (1 mg) was placed in a microcentrifuge tube containing RIPA Lysis buffer (Applygen, Beijing, China) and homogenized on ice. After centrifugation, the supernatant was transferred to a new tube, and protein concentration was determined using the BCA Protein Assay (Applygen, Beijing, China). After lysing, 18 μg of protein was loaded onto and resolved by SDS-PAGE electrophoresis (Bio-Rad, USA) and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membranes were blocked with 5% nonfat milk in PBS-Tween 20 (PBS-T) for 2 hours at room temperature, and the membranes were incubated overnight with the specific primary antibodies against TGF-β1 (rabbit anti-rat TGF-β1 monoclonal antibody, 1 : 1,000, AbCam, UK), Smad7 (rabbit anti-rat, Smad7 monoclonal antibody 1 : 500, Sigma, USA), or GAPDH (rabbit anti-rat GAPDH monoclonal antibody 1 : 2,000, Beyotime, China), respectively. On the next day, the membranes were washed with PBS-T three times and then incubated with sheep anti-rabbit secondary antibody conjugated with HRP (Antgene, Wuhan, China) for 1 hour at room temperature. The specific protein bands were detected with chemiluminescence in a darkroom using BeyoECL Plus (ECL Chemiluminescence Kit, ASPEN, WuHan, China) and documented and analyzed using Image Acquisition and Analysis Software (Flour Chem FC3 software, Alpha Innotech, USA).
Bca protein assay
Manufactured by Applygen
Sourced in China
The BCA protein assay is a laboratory tool used to quantify the total protein concentration in a sample. It utilizes the bicinchoninic acid (BCA) method, which involves the reduction of copper ions to produce a purple-colored complex that can be measured spectrophotometrically. The assay provides a simple and reliable way to determine protein levels in various biological and chemical samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Nuclear and cytoplasmic protein extraction kit, by Beyotime (2 mentions)
Lysis buffer, by Solarbio (2 mentions)
Fusion fx5 spectra, by Vilber (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Sds page electrophoresis, by Bio-Rad (1 mentions)
Rabbit antirat tgf β1 monoclonal antibody, by Abcam (1 mentions)
Ripa lysis buffer, by Applygen (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Donkey anti goat igg hrp sc 2020, by Santa Cruz Biotechnology (1 mentions)
Western lightning chemiluminescence reagent plus, by PerkinElmer (1 mentions)
Anti β actin sc 1615, by Santa Cruz Biotechnology (1 mentions)
Anti hsp70 sc 66048, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse igg hrp sc 2005, by Santa Cruz Biotechnology (1 mentions)
Gapdh antibody, by Merck Group (1 mentions)
Hyperfilm, by Bio-Rad (1 mentions)
Anti total erk1 2 antibody, by Cell Signaling Technology (1 mentions)
Anti mfn2, by Santa Cruz Biotechnology (1 mentions)
Anti eif 5, by Santa Cruz Biotechnology (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
12 protocols using bca protein assay
1
Western Blot Analysis of TGF-β1 and Smad7
2
Lipid and Inflammation Biomarkers Analysis
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Lipid markers including TC, TG, HDL-c, LDL-c, apolipoprotein AI (ApoAI), apolipoprotein B (ApoB) and high sensitivity C-reactive protein (HsCRP) were measured by a biochemistry analyzer in the clinical laboratory centre of Fuwai hospital, as described previously [21 (link)]. And the plasma apoCIII concentrations and HDL-apoCIII were measured by using an ELISA kit (Abcam, UK). The concentrations of HDL samples were quantified by using BCA protein assay (Applygen Technologies Inc, China). The effect of the differences of HDL sample concentrations was considered, and all the ELISA results (ng/ml) of HDL-apoCIII were adjusted by the concentrations (ug/ml) of HDL samples, and the final HDL-apoCIII unit was reported in ug/mgHDL.
3
Tissue Homogenization and Protein Extraction
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The NAc tissues (both the core and shell included) were homogenized using a mixture of RIPA lysis buffer. The homogenates were centrifuged at 12,000× g for 20 min at 4 °C after being incubated for 60 min on ice. Using a BCA protein assay (Applygen Technologies Inc, Beijing, China), the protein concentrations in the supernatants were determined after their collection. For future usage, all of the protein homogenates were kept at −80 °C.
4
Western Blot Analysis of TLR4 Signaling
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The heart tissues were homogenized, and the lysates were collected to obtain the total protein using lysis buffer (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China). Nuclear proteins were extracted using a nuclear and cytoplasmic protein extraction kit (Beyotime Biotechnology Co., Ltd., Beijing, China), according to the manufacturer’s protocol. Total protein and nuclear protein were extracted and their concentrations in all samples were quantified with the bicinchoninic acid (BCA) protein assay (Applygen Technologies Inc., Beijing, China). Subsequently, proteins were separated on 10% SDS-PAGE gel and transferred to a PVDF membrane. After blocking in 5% skimmed milk for 2 h, the membranes were incubated with primary antibodies against TLR4 (1:1000), NF-κB (1:1000), IL-6 (1:1000), TAK-1 (1:750), GAPDH (1:10000) and H3 (1:10000). Then the membranes were incubated with an HRP-conjugated secondary antibody followed by ECL detection (Vilber Fusion FX5 Spectra, Paris, France). And the bands were analyzed semi-quantitatively with Image J software (National Institutes of Health, Bethesda, Maryland, USA).
5
Western Blot Analysis of Lung Tissue Proteins
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Mice lung tissues were lysed in RIPA buffer (Beyotime, Haimen, Jiangsu, China) with 10 mM PMSF (Beyotime, Haimen, Jiangsu, China) and 20 mM Cocktail (Roche) on ice for 20 min. The lysates were then centrifuged at 4°C for 5 min at 10,000 g in a microcentrifuge to remove cell debris. Proteins were extracted from the supernatant. Total protein [30 μg; as determined by BCA protein assay (Applygen, Beijing, China)] was boiled for 5 min and resolved in 12% polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The blotted membranes were incubated in 5% skim milk to block nonspecific absorption of antibodies. The membranes were then immunoblotted with the following antibodies and dilutions: anti-HSP 70 (SC-66048) 1:200, anti-β-actin (SC-1615) 1:5000, goat anti-mouse IgG-HRP (sc-2005) 1:2000, and donkey anti-goat IgG-HRP (SC-2020) 1:5000 (each Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Proteins were detected using Western Lightning Chemiluminescence Reagent Plus (Perkin–Elmer Life Sciences, Inc., Norwalk, CT, USA) and visualized by exposure to Fuji medical X-ray film (RX-U; Fuji Photo Film Co., Tokyo, Japan).
6
Quantification of Spinal Cord MCP1 and CCR2 Proteins
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Spinal cord tissues were collected from deeply anesthetized animals perfusing transcardially with 50 mL of 0.9% NaCl on day 14. Protein concentrations were determined by BCA Protein Assay (Applygen, Beijing, China). Proteins (50 μg) were loaded for each lane and separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proteins were then transferred and incubated overnight at 4 °C with Anti-MCP1 (CCL2) antibody (ab25124) (1:1000, mouse; Abcam, Cambrige, MA, USA.), CCR2 antibody (36784) (mouse, 1:1000, mouse; Signalway Antibody LLC, College Park, MD, USA.); GAPDH antibody (1:10,000, mouse; Sigma-Aldrich Co. LLC., (St. Louis, MO, USA), was used as loading control. These blots were further incubated with horseradish peroxidase–conjugated secondary antibody, developed in enhanced chemiluminescence solution, and exposed on Hyperfilm (Bio-Rad, Hercules, CA, USA,) for 1 to 5 min. Specific bands were evaluated by apparent molecular size. The intensity of the selected bands was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Cellular proteins were extracted from the primary microglial cells and astrocytes using RIPA buffer. The homogenates were centrifuged for 15 min at 12,000× g at 4 °C. The quantity of protein in each supernatant was determined using a BCA Protein Assay kit and then processed as shown above.
7
Western Blot Analysis of Mfn2 and ERK1/2
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Total protein was extracted in RIPA buffer (CxBio, Shanghai, China) supplemented with phenylmethanesulfonyl (PMSF, CxBio). The protein concentrations were measured using the BCA Protein Assay (Applygen, Beijing, China). The samples were separated on a 10% SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane, which was blocked in 5% skim milk for 1 h and then incubated with primary antibodies at 4 °C overnight. After the washing steps, the membranes were incubated with horseradish peroxidase-labelled secondary antibodies for 1 h at room temperature. The bands were visualized using a chemiluminescence detection system, and the densitometric results were analysed with Image J software. EIF5 levels were used as internal controls for protein normalization.
Anti-Mfn2 and anti-EIF5 for western blotting were purchased from Santa Cruz Biotechnology (CA, USA), and anti-phospho-ERK1/2 and anti-total-ERK1/2 antibodies were purchased from Cell Signalling Technology (Beverly, MA, USA).
Anti-Mfn2 and anti-EIF5 for western blotting were purchased from Santa Cruz Biotechnology (CA, USA), and anti-phospho-ERK1/2 and anti-total-ERK1/2 antibodies were purchased from Cell Signalling Technology (Beverly, MA, USA).
8
Western Blot Analysis of Liver and Aorta Proteins
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Liver or aorta tissues were lysed in sample buffer containing 62 mM Tris–HCl, pH 6.8, 0.1% SDS, 0.1 mM sodium orthovanadate, and 50 mM sodium fluoride. The protein content was determined by the BCA protein assay (Applygen Technologies Inc., Beijing, China). Equal amount of proteins was loaded and separated by SDS-PAGE. After electrophoresis, the proteins were transferred on membranes, after being blocked with 3% non-fat dry milk, the membrane with target proteins was recognized with primary antibodies against CD36, SR-A, SR-BI, PPARα, ABCA1, ABCG1, ABCG5, ABCG8, and GAPDH (Abcam, Cambridge, MA, United States). The bands were detected using an ECL detection kit (Applygen Technologies, Beijing, China). For quantification, band intensity was assessed by densitometry and expressed as mean area density using Quantity One image analyzer software (Bio-Rad, Richmond, CA, United States).
9
Protein Extraction and Western Blotting
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Tissues and cultured cells were lysed in RIPA buffer (Beyotime) containing protein inhibitors (Selleck). The protein concentration was determined by a BCA protein assay (Applygen Technologies Inc.). Proteins were mixed with 5× SDS sample loading buffer and incubated at 95°C for 10 min. Then, proteins were separated by SDS–PAGE gel, transferred to PVDF membranes (Millipore), and incubated with appropriate primary antibodies coupled with a horseradish peroxidase-conjugated secondary antibody. Images were visualized using ECL western blotting detection reagents (Tanon).
10
Western Blot Analysis of Smad Proteins
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The lysates were clarified by centrifugation and the supernatants collected. Protein concentrations were determined using the bicinchoninic acid assay (BCA) Protein Assay (Applygen, Beijing, China). Equivalent amounts of tissue protein (80 μg) were resolved on SDS polyacrylamide gels, and transferred by electroblotting to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% (W/V) nonfat milk at room temperature for 1 h, and then incubated overnight at 4°C with the primary antibody against Smad3 (dilution 1: 200, Santa Cruz, CA, USA), Smad7 (dilution 1:200, Santa Cruz, CA, USA), p-Smad3 (dilution 1:1000, Epitomics, CA, USA), and β-actin (dilution 1: 1000, Santa Cruz, CA, USA). After washing in a buffer containing Tris-buffered saline (TBS), with 0.1% Tween, the membranes were incubated with horseradish peroxidase (HRP)-linked anti-mouse secondary antibody at a dilution of 1:3000. Following washing in 0.1% Tween TBS buffer, the immunolabeled proteins were detected by enhanced chemiluminescense reagents (Applygen, Beijing, China). The density of the detected bands was analyzed by Quantity One software.
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