The effective concentrations of siULL1 and JNK inhibitor were selected for exploring the role of UFL1 in the process of milk protein and fat synthesis. siUFL1 was transfected by Lipofectamine 2000 reagent. The concentration of 20 μM of SP600125 used in the starvation medium for 2 h suppressed the JNK expression. After SP600125 treatment, proteins from the treated samples were collected and analyzed by western blotting. Cells were lysed in 200 μL lysis buffer containing RIPA buffer (P0013B; Beyotime Biotechnology) and PMSF buffer (ST506, Beyotime Biotechnology). Protein concentrations were determined using the BSA assay kit (P0010; Beyotime Biotechnology). Equal amounts of protein samples (30–60 μg) were separated using 12% SDS-PAGE and electrotransferred onto PVDF membranes (FFP39, Beyotime Biotechnology). Membranes were blocked with 5% free-fat milk for 2 h at room temperature and subsequently incubated with primary antibodies at 4°C overnight (Table 1 ). After three washes with PBS, membranes were subsequently rinsed with TBST for 5 min and incubated with secondary antibodies for 2 h at room temperature. The membranes were visualized using LAS-4000 (Fujifilm, Tokyo, Japan).
Bsa assay kit
Manufactured by Beyotime
Sourced in China
The BSA assay kit is a laboratory tool used for the quantitative determination of protein concentration. It provides a standardized method to measure the amount of bovine serum albumin (BSA) present in a sample. The kit contains necessary reagents and a protocol to perform the assay.
Automatically generated - may contain errors
Lab products found in correlation
Ffp39, by Beyotime (1 mentions)
Pmsf buffer, by Beyotime (1 mentions)
Las 4000, by Fujifilm (1 mentions)
Amersham ecl plus, by Cytiva (1 mentions)
Ab16663, by Abcam (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Ecl reagent, by Beyotime (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Beyoecl kit, by Beyotime (1 mentions)
Anti transgelin, by Abcam (1 mentions)
Anti cofilin 1, by Abcam (1 mentions)
Anti gelsolin, by Abcam (1 mentions)
Anti hsp27, by Abcam (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Gel pro analyzer software, by Media Cybernetics (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β actin, by Boster Bio (1 mentions)
5 protocols using bsa assay kit
1
Regulation of Milk Protein and Fat Synthesis
2
Protein Expression Analysis in HepG-2 Cells
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5 mL of HepG-2 cells (1 × 105 cells per mL) were seeded on a 100 mm diameter dish and incubated for 24 h at 37 °C, in 5% CO2/95% air atmosphere. Cells were harvested and washed 3 times with ice-cold PBS after treatment of L6 (5 μM) or C6 (5 μM) for 24 hours. The cells were disrupted with buffer and centrifuged at 10 000g for 15 minutes under 4 °C to obtain a supernatant. The protein concentration was determined using a BSA Assay Kit (Beyotime, China), and the samples were detected by SDS-polyacrylamide gel electrophoresis. Under ice bath conditions, the protein was loaded on the polyvinylidene fluoride membrane for 3 hours, followed by preparation of skim milk (5%) for the presence of protein-blocked 1 h in TBST buffer at 25 °C. Membranes and primary antibodies were incubated overnight at 4 °C and washed three times with TBST buffer (20 mM Tris–HCl, 0.05% Tween 20, 150 mM NaCl, pH 8.0). After the membranes were incubated with the secondary antibody for 1 h at 25 °C, they were washed three times in the same manner. Immunoreactivity was imaged by Amersham ECL Plus (Amersham) and the amount of protein in each lane was determined by β-actin.
3
Protein Expression Analysis in Cells
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Total protein was extracted using RIPA buffer (Beyotime, Shanghai, China). The concentrations of total proteins were determined using BSA assay kit (Beyotime). Followingly, proteins were separated on 10-20% SDS gel and transferred to a PVDF membrane. The blots were incubated with primary antibody against IL-6 (#66146-1-Ig, 1:1000, ProteinTech, USA), STAT3 (#10253-2-AP, 1:1000, ProteinTech, USA), p-STAT3 (#9131L, 1:500, Cell Signaling, USA), Bcl2 (#12789-1-AP, 1:1000, ProteinTech, USA), CyclinD1 (#ab16663, 1:1000, Abcam), β-actin (#ab8226, 1:1000, Abcam). The secondary IgG-HRP antibodies were used and protein bands were determined using ECL reagent (Beyotime). The bands were quantified using Image J software (NIH, USA).
4
Proteomic Analysis of Aortic Tissue Lysates
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Aorta segment lysis was performed with chilled lysis buffer in presence of protease inhibitors. Lysate centrifugation was carried out at 12,000 rpm for 15 min at 4 °C. Total protein in the supernatant was quantitated with a BSA assay kit (P0006, Beyotime, Jiangsu, China). Protein separation was performed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and protein bands were electro-transferred onto polyvinylidene difluoride (PVDF) membranes. The samples were incubated overnight at 4 °C with anti-tropomyosin β, anti-transgelin, anti-annexin, anti-gelsolin, anti-HSP-27, anti-cofilin-1 and anti-GAPDH (1:1000; Abcam, Cambridge, UK) primary antibodies. Further incubation was performed with goat anti-mouse or anti-rabbit secondary antibodies (1:10,000; Santa Cruz Biotechnology, USA) for 1h in ambient conditions. Development was carried out with the BeyoECL kit (Beyotime, China) and a Tanon 5200 system.
5
Microsomal Preparation and Western Blot Analysis
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For the microsomal preparation, the liver homogenate was centrifuged at 9000× g for 20 min at 4 °C, and the supernatant was transferred to a tube with 88 mM CaCl 2 , after being shaken on ice for 5 min, the mixture was centrifuged at 27000× g for 20 min at 4 °C. The pellets were collected and resuspended in 50 mM Tris-HCl mixed with 20% glycerol. The protein concentration was determined using a BSA assay kit (Beyotime Institute of Biotechnology, Beijing, China).
Protein samples were extracted from liver tissue and separated by SDS-PAGE. After being transferred to PVDF membranes (Millipore Corporation, Boston, MA, USA), the membranes were blocked in TBST containing 5% skimmed milk powder and incubated overnight at 4 °C with primary antibodies (CYP2E1, 1 : 500, Cat. No. BA1774-2, Boster, Wuhan, China; β-actin 1 : 1000, Cat. No. LK9001T, Sungene Biotech, Tianjin, China). After washing three times with TBST, the membranes were incubated with horseradish peroxidase conjugated secondary antibodies (1 : 2000, Cat. No. LK2001, Sungene Biotech, Tianjin, China) for 2 h at room temperature. Protein bands were visualized by ECL reaction (Genshare Biological, Xi'an, China) and the protein levels were quantified using Gel-Pro Analyzer software (Media Cybernetics, Bethesda, USA) as normalized to beta-actin.
Protein samples were extracted from liver tissue and separated by SDS-PAGE. After being transferred to PVDF membranes (Millipore Corporation, Boston, MA, USA), the membranes were blocked in TBST containing 5% skimmed milk powder and incubated overnight at 4 °C with primary antibodies (CYP2E1, 1 : 500, Cat. No. BA1774-2, Boster, Wuhan, China; β-actin 1 : 1000, Cat. No. LK9001T, Sungene Biotech, Tianjin, China). After washing three times with TBST, the membranes were incubated with horseradish peroxidase conjugated secondary antibodies (1 : 2000, Cat. No. LK2001, Sungene Biotech, Tianjin, China) for 2 h at room temperature. Protein bands were visualized by ECL reaction (Genshare Biological, Xi'an, China) and the protein levels were quantified using Gel-Pro Analyzer software (Media Cybernetics, Bethesda, USA) as normalized to beta-actin.
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