Total protein was isolated using a total protein isolation kit (Solarbio, China) and its concentrations were estimated with the BCA method (Solarbio, China). Proteins (30 μg/per well) were applied for electrophoresis (5%∼10% SDS-PAGE) and then transferred onto polyvinylidene difluoride membranes (0.2 µM, Bio-Rad, USA). Subsequently, the PVDF membrane was blocked for 1.5 h at room temperature and then incubated with primary antibodies against iNOS (1:1000, ab178945), Arg1 (1:1000, CST93668), SIRT1 (1:1000, ab189494), acetyl-NF-κB p65 (Lys310) (1:1000, CST3045), phospho-NF-κB p65 (Ser536) (1:1000, CST3033), NF-κB-p65 (1:1000, CST8242), β-actin (1:2000, ab8226) overnight at 4 °C, and then incubated with secondary antibodies (1:3000, CST7074) for 1 h. Enhanced chemiluminescence (170-5070, Bio-Rad, USA) was applied for blots developing and the density was calculated by ImageJ system.
Total protein isolation kit
Manufactured by Solarbio
Sourced in China
The Total protein isolation kit is a laboratory equipment designed to extract and purify total protein from various biological samples. It provides a simple and efficient method to obtain high-quality protein samples for further analysis and experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 1000, by Thermo Fisher Scientific (5 mentions)
Primary anti β actin antibody, by Wuhan Boster Biological Technology (3 mentions)
Rabbit anti hmgb1 polyclonal antibody, by Abcam (3 mentions)
Genetools software, by Syngene (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Bca method, by Solarbio (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Enhanced chemiluminescence western blot detection kit, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Polyvinylidene uoride membrane, by Merck Group (1 mentions)
5 protocols using total protein isolation kit
1
Protein Isolation and Western Blot Analysis
2
Western Blot Analysis of Lung Proteins
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The proteins from right lung tissues were extracted using a total protein isolation kit (Solarbio, China), the concentrations of protein were quantified by the BCA protein assay kit (Solarbio, China), and 30 μg of protein was loaded per well on 10% SDS-PAGE for electrophoresis. Subsequently, proteins were transferred to PVDF membranes (Bio-Rad, USA) and blocked with 5% skimmed milk in TBST for 3 h at room temperature. Then, membranes were incubated at 4 °C overnight with primary antibodies for HO-1 (1:1000, CST5061S), MTCO1(1:2000, ab14705), SDHA (1:2000, ab14715), HSP60 (1:1000, ab190828), LONP1 (1:1000, ab224316), HSP90 (1:2000, ab13492), PPARγ (1:1000, ab178860), PGC1α (1:2000, ab106814), and β-actin (1:3000, AF7018). After washing five times with TBST, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibody (1:3000, S0001) for 1 h at room temperature. Proteins were visualized by an enhanced chemiluminescence Western blot detection kit (170-5070, Bio-Rad, USA). The relative expression of target proteins was quantified by the ImageJ-Analysis system, and β-actin served as a standard reference.
3
HMGB1 Protein Quantification Protocol
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Tissue total proteins were extracted using the total protein isolation kit (Solarbio, Beijing, China), according to manufacturers' instructions. Proteins were quantified using the Nanodrop 1000 (Thermo Scientific, Wilmington, Del.). Samples were denatured at 100°C for five minutes. Equal protein concentrations were loaded onto 12% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (Millipore Corporation, Billerica, Mass.). Membranes containing proteins were blocked in 5%
BSA and incubated with rabbit anti-HMGB1 polyclonal antibody (1:1,000, Abcam, U.S.A), followed by incubation with horseradish peroxidase-linked secondary antibodies (1:5,000; Golden Bridge Biotech Co. Ltd, Beijing, China). For standardization and expression comparisons, the membranes were also hybridized with a primary anti-β-actin antibody (1:1,000; Wuhan Boster Biological Technology, Wuhan, China). The bands appearing on film were analyzed with GeneTools software (Syngene, Frederick, Md).
BSA and incubated with rabbit anti-HMGB1 polyclonal antibody (1:1,000, Abcam, U.S.A), followed by incubation with horseradish peroxidase-linked secondary antibodies (1:5,000; Golden Bridge Biotech Co. Ltd, Beijing, China). For standardization and expression comparisons, the membranes were also hybridized with a primary anti-β-actin antibody (1:1,000; Wuhan Boster Biological Technology, Wuhan, China). The bands appearing on film were analyzed with GeneTools software (Syngene, Frederick, Md).
4
Western Blot Analysis of HMGB1 Protein
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Tissue total proteins were extracted using the total protein isolation kit (Solarbio, Beijing, China), according to manufacturers' instructions. Proteins were quanti ed using the Nanodrop 1000 (Thermo Scienti c, Wilmington, Del.).
Samples were denatured at 100 °C for ve minutes. Equal protein concentrations were loaded onto 12% SDS-PAGE gels and transferred to polyvinylidene uoride membranes (Millipore Corporation, Billerica, Mass.). Membranes containing proteins were blocked in 5% BSA and incubated with rabbit anti-HMGB1 polyclonal antibody (1:1,000, Abcam, U.S.A), followed by incubation with horseradish peroxidase-linked secondary antibodies (1:5,000; Golden Bridge Biotech Co. Ltd, Beijing, China).
For standardization and expression comparisons, the membranes were also hybridized with a primary anti-β-actin antibody (1:1,000; Wuhan Boster Biological Technology, Wuhan, China). The bands appearing on lm were analyzed with GeneTools software (Syngene, Frederick, Md).
Samples were denatured at 100 °C for ve minutes. Equal protein concentrations were loaded onto 12% SDS-PAGE gels and transferred to polyvinylidene uoride membranes (Millipore Corporation, Billerica, Mass.). Membranes containing proteins were blocked in 5% BSA and incubated with rabbit anti-HMGB1 polyclonal antibody (1:1,000, Abcam, U.S.A), followed by incubation with horseradish peroxidase-linked secondary antibodies (1:5,000; Golden Bridge Biotech Co. Ltd, Beijing, China).
For standardization and expression comparisons, the membranes were also hybridized with a primary anti-β-actin antibody (1:1,000; Wuhan Boster Biological Technology, Wuhan, China). The bands appearing on lm were analyzed with GeneTools software (Syngene, Frederick, Md).
5
HMGB1 Protein Quantification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Tissue total proteins were extracted using the total protein isolation kit (Solarbio, Beijing, China), according to manufacturers' instructions. Proteins were quantified using the Nanodrop 1000 (Thermo Scientific, Wilmington, Del.). Samples were denatured at 100°C for five minutes. Equal protein concentrations were loaded onto 12% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (Millipore Corporation, Billerica, Mass.). Membranes containing proteins were blocked in 5%
BSA and incubated with rabbit anti-HMGB1 polyclonal antibody (1:1,000, Abcam, U.S.A), followed by incubation with horseradish peroxidase-linked secondary antibodies (1:5,000; Golden Bridge Biotech Co. Ltd, Beijing, China). For standardization and expression comparisons, the membranes were also hybridized with a primary anti-β-actin antibody (1:1,000; Wuhan Boster Biological Technology, Wuhan, China). The bands appearing on film were analyzed with GeneTools software (Syngene, Frederick, Md).
BSA and incubated with rabbit anti-HMGB1 polyclonal antibody (1:1,000, Abcam, U.S.A), followed by incubation with horseradish peroxidase-linked secondary antibodies (1:5,000; Golden Bridge Biotech Co. Ltd, Beijing, China). For standardization and expression comparisons, the membranes were also hybridized with a primary anti-β-actin antibody (1:1,000; Wuhan Boster Biological Technology, Wuhan, China). The bands appearing on film were analyzed with GeneTools software (Syngene, Frederick, Md).
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