Proteins were extract from brain homogenates or cultured cells with T-Per or M-Per reagents, respectively. Equal amounts of proteins were loaded and separated on a 4%−15% Criterion Precast Midi Protein Gel (Bio-Rad) and then transferred to a 0.22 mm pore size PVDF membrane. After blocking with 5% non-fat milk in TBST and washing for 3 times, the membrane was immunoblotted with the primary antibody overnight at 4 °C and with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (Vector Laboratories). The bands were visualized by Pierce SuperSignal Substrate or Femto Maximum Sensitivity Substrate, and the images were captured by ChemiDoc Imaging System. Band intensities were quantified using Image Lab 6.0.1.
Hrp conjugated anti rabbit or anti mouse secondary antibody
Manufactured by Vector Laboratories
HRP-conjugated anti-rabbit or anti-mouse secondary antibodies are laboratory reagents used to detect and visualize target proteins in various immunoassays, such as Western blotting and immunohistochemistry. These antibodies are conjugated with the enzyme horseradish peroxidase (HRP), which can catalyze a colorimetric or chemiluminescent reaction for signal detection.
Automatically generated - may contain errors
Lab products found in correlation
Criterion precast midi protein gel, by Bio-Rad (3 mentions)
Image lab 6, by Bio-Rad (2 mentions)
Protein assay dye reagent, by Bio-Rad (2 mentions)
Pierce supersignal chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Horseradish peroxidase, by Vector Laboratories (1 mentions)
Chemiluminescent detection kit ecl plus, by Cytiva (1 mentions)
Luminescent image analyzer, by Fujifilm (1 mentions)
4 protocols using hrp conjugated anti rabbit or anti mouse secondary antibody
1
Western Blot Protein Analysis
2
Protein Extraction and Western Blot Analysis
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Tissues were lysed in the urea lysis buffer (25 mM Tris-HCl, 8 M Urea, 1% SDS, 1 mM EDTA, 0.7 M DTT, pH 7.4). After ultrasonic homogenization, centrifugation was performed at 12,000 × g for 15 min. The whole lysate was separated by 8–15% gradient SDS-PAGE gel electrophoresis and transferred onto the PVDF membrane. The membranes were incubated with the primary antibodies (Supplementary Table 1 ), probed with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibodies (1:5000, Vector Laboratories), developed using a Chemiluminescent Detection Kit (ECL Plus, Amersham Pharmacia Biotech), and analyzed using a Luminescent Image Analyzer (las-4000mini, Fujifilm). The target protein was then normalized to β-actin as the loading control.
3
Western Blot Quantification of Neuronal and Astrocytic Proteins
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Protein concentrations of cell lysate of neurons and astrocytes were determined by Protein Assay Dye Reagent (Bio-Rad). Equal amounts of proteins (10–20 μg) were loaded and separated on a 4%–15% Criterion Precast Midi Protein Gel (Bio-Rad) and then transferred to a 0.45 μm pore size polyvinylidene difluoride (PVDF) membrane and immunoblotted with the appropriate primary antibodies. Primary antibodies were incubated overnight, followed by washing and probing with the appropriate HRP-conjugated anti-rabbit or anti-mouse secondary antibody (Vector Laboratories, Burlingame, CA). The immunoreactive bands were visualized by Pierce SuperSignal Chemiluminescent Substrate or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, IL) and captured by ChemiDoc Imaging System (BioRad, Hercules, CA). Saturated pixel highlighting feature of the instrument was used to prevent image overexposure for chemiluminescence. Band intensity was quantified using Image Lab 6.0.1 (Bio-Rad, Hercules, CA).
4
Western Blot Quantification of Neuronal and Astrocytic Proteins
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