Liver tissues were homogenized and treated with RIPA lysis buffer (1 mM PMSF, 5 mM EDTA, 1 mM sodium orthovanadate, 150 mM sodium chloride, 8 μg/mL leupeptin, and 1.5% Nonidet P-40, and 20 mM Tris–HCl, pH 7.4) with phosphatase and protease inhibitors. The samples (30 μg of proteins) were separated in 4–15% polyacrylamide gel and then transferred to PVDF membrane (0.2 mM) by Trans-Blot Turbo (BioRad, Hercules, CA, USA). Nonspecific binding sites on the blots were blocked by incubation in 5% BSA for 1 h, and the membranes were then incubated overnight with primary antibodies and incubated with secondary antibodies for 1 h. Immune complexes were detected by the ECL chemiluminescence system (Amersham Pharmacia, Little Chalfont, UK), according to the manufacturer’s instructions. The primary antibodies used were anti-LPS (Abcam) and anti-β-actin antibody (1:20,000; Sigma). The secondary antibodies used were HRP-conjugated anti-rabbit (Abcam) and anti-mouse antibodies (Abcam). The chemiluminescent blots were acquired by Chemidoc and analyzed using ImageJ software. The protein expression levels were standardized relative to the level of β-actin.
Anti mouse antibodies
Manufactured by Abcam
Anti-mouse antibodies are used in various laboratory applications to detect and quantify mouse proteins or other target molecules. They can bind specifically to mouse antigens and facilitate their identification and measurement.
Automatically generated - may contain errors
2 protocols using anti mouse antibodies
1
Liver Protein Immunoblotting Analysis
2
Protein Quantification and Oxidative Stress Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Renal tissues were homogenized in lysis buffer (1% Triton X-100, 150 mmol/L NaCl,
50 mmol/L Tris, pH 8.0, 1 mmol/L EDTA, 10 mg/L phenylmethylsulfonyl fluoride).
Protein concentration was determined by the Bradford assay. Equal amounts (50 μg
protein/lane) of proteins were loaded onto 12% sodium dodecyl sulfate
polyacrylamide gel electrophoresis, electrophoresed, and transferred to
polyvinyl difluoride membranes. Membranes were blocked with a 5% nonfat milk for
1 hour and then incubated with antimouse antibodies (Abcam, Cambridge,
Massachusetts) against 3-nitrotyrosine (3-NT at 1:1400 dilution),
4-hydroxynonenal (4-HNE,1:50), collagen IV (Col IV,1:500), and fibronectin
(FN,1:5000) overnight at 4°C. Membranes were washed to remove the unbound
antibodies with Tris-buffered saline (pH 7.2), containing 0.05% Tween 20 3 times
and then incubated with the appropriate secondary antibody. Antigen–antibody
complexes were visualized by electrochemiluminisence, and the resulting image
was analyzed with Bio-Rad Quantity One (Hercules, CA).
50 mmol/L Tris, pH 8.0, 1 mmol/L EDTA, 10 mg/L phenylmethylsulfonyl fluoride).
Protein concentration was determined by the Bradford assay. Equal amounts (50 μg
protein/lane) of proteins were loaded onto 12% sodium dodecyl sulfate
polyacrylamide gel electrophoresis, electrophoresed, and transferred to
polyvinyl difluoride membranes. Membranes were blocked with a 5% nonfat milk for
1 hour and then incubated with antimouse antibodies (Abcam, Cambridge,
Massachusetts) against 3-nitrotyrosine (3-NT at 1:1400 dilution),
4-hydroxynonenal (4-HNE,1:50), collagen IV (Col IV,1:500), and fibronectin
(FN,1:5000) overnight at 4°C. Membranes were washed to remove the unbound
antibodies with Tris-buffered saline (pH 7.2), containing 0.05% Tween 20 3 times
and then incubated with the appropriate secondary antibody. Antigen–antibody
complexes were visualized by electrochemiluminisence, and the resulting image
was analyzed with Bio-Rad Quantity One (Hercules, CA).
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