The tissues were lysed utilizing RIPA buffer (1 ml/100 mg) at 0°C, and centrifuged at a speed of 12,000 × g for 10 min. The total protein concentration was determined using a BCA assay kit (cat. no. 23227; Pierce Biotechnology, Rockford, Illinois, USA) according to the instructions of the manufacturer. The total protein (40 µg) was loaded for 15% SDS-PAGE electrophoresis, and then the proteins were transferred to PVDF membranes. The PVDF membranes were blocked with 5% non-fat dry milk at 37°C for 1 h, and then incubated with anti-rat AQP4 (cat. no. ab156924; 1:2,000; Abcam) or anti-rat β-actin (cat. no. ab8227; 1:2,000; Abcam) antibody at 4°C for 12 h. Finally, the membranes were incubated with HRP-labeled goat anti-mouse IgG antibody (cat. no. ab6789; 1:1,000; Abcam) at 37°C for 1 h. The immunoreactivity of the bands was detected using an enhanced chemiluminescence reagent (ECL; Amersham, Piscataway, New Jersey, USA) on X-ray film. The densities of AQP4 and β-actin were quantified using a Gel Image Analysis System (Labworks 4.6; UVP LLC, Upland, CA, USA). The relative AQP4 expression was normalized to those of β-actin.
Hrp labeled goat anti mouse igg antibody
Manufactured by Abcam
Sourced in United Kingdom
The HRP-labeled goat anti-mouse IgG antibody is a secondary antibody used for the detection of mouse primary antibodies in various immunoassays. It is conjugated with horseradish peroxidase (HRP), an enzyme that can be used as a reporter for colorimetric or chemiluminescent detection.
Automatically generated - may contain errors
2 protocols using hrp labeled goat anti mouse igg antibody
1
Western Blot Analysis of Aquaporin-4 Expression
2
Screening Hybridomas for Abrin-Specific Antibodies
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Indirect ELISAs were employed to screen the established hybridomas. Briefly, microwell plates were coated with 100 µL of 5 µg/mL abrin protein in 0.05 M bicarbonate buffer (pH 9.6) at 4 °C overnight and blocked with 300 µL of 1% (m/v) BSA for 2 h at 37 °C. After the blocking steps, 100 µL of supernatant from the hybridoma cultures serially diluted in PBST containing 0.5% BSA was added to the wells to incubate for 1 h at room temperature. After the wells were washed three times with PBST, bound antibodies were detected with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody (Abcam, Cambridge, UK). Following washing, the colorimetric reaction was measured using 3,3′,5,5′-tetramethylbenzidine (TMB) peroxidase substrate after a 10 min development period. The reaction was then terminated with 2 M H2SO4 (Sigma-Aldrich, St. Louis, MO, USA), and the absorbance at 450 nm was measured using a microplate reader (Infinite M1000 pro, TECAN, Männedorf, Switzerland).
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