For protein extraction, cells or tissues were manually homogenized in Hunt buffer [20 mM tris-HCl (pH 8.0), 100 mM sodium chloride, 1 mM EDTA, and 0.5% NP-40] supplemented with Halt protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific). After full-speed centrifugation, the supernatant containing the soluble protein fraction was further used. Equal amounts of 20 to 30 μg of protein were separated by SDS–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Immobilion-P, Merck Millipore) according to standard protocols. Blots were blocked for 1 hour with 5% milk in TBST [1× tris-buffered saline (TBS) and 0.1% Tween 20] and were then incubated overnight at 4°C with primary antibodies diluted in 5% milk in TBST. Blots were washed three times in TBST for 5 min and further incubated with horseradish peroxidase–conjugated secondary anti-mouse–IgG–H&L chain (Promega) or anti-rabbit-IgG-F(ab′)2 (GE Healthcare) antibody for 1 hour at room temperature, washed three times in TBST for 5 min, and visualized using enhanced chemiluminescence (ECL, GE Healthcare). See table S1 for a list of antibodies used in this study. β-Actin was used to control for protein loading.
Anti rabbit igg f ab 2
Manufactured by GE Healthcare
The Anti-rabbit-IgG-F(ab′)2 is a laboratory reagent used for immunological detection and quantification applications. It is a fragment derived from rabbit immunoglobulin G (IgG) that specifically binds to the Fab region of the target antibody, allowing for targeted detection or purification.
Automatically generated - may contain errors
3 protocols using anti rabbit igg f ab 2
1
Protein Extraction and Western Blotting
2
Protein Extraction and Western Blot Analysis
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For protein extraction, cells or tissues were manually homogenized in Hunt buffer (20 mM Tris-HCl, pH 8.0, 100 mM sodium chloride, 1 mM EDTA, and 0.5% NP-40) supplemented with Halt protease/phosphatase inhibitor cocktail (ThermoFisher Scientific). After full-speed centrifugation, the supernatant containing the soluble protein fraction was further used. Equal amounts of 20–30 µg of proteins were separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Immobilion-P, Merck Millipore) according to standard protocols. Blots were blocked for 1 h with 5% milk in TBST (1× Tris-buffered saline and 0.1% Tween-20) and were then incubated overnight at 4°C with primary antibodies diluted in 5% milk in TBST. Blots were washed three times in TBST for 5 min and were then incubated with HRP-conjugated secondary anti-mouse-IgG-H&L chain (Promega) or anti-rabbit-IgG-F(ab′)2 (GE Healthcare) antibody for 1 h at room temperature, washed three times in TBST for 5 min, and visualized using enhanced chemiluminescence (GE Healthcare). See Table S2 for a list of antibodies used in this study. β-Actin was used to control for protein loading. IgM was detected by incubating blots for 1 h with HRP-conjugated secondary anti-mouse-IgG-H&L chain (GE Healthcare).
3
Chondroitin Sulfate Proteoglycan Analysis
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Hippocampi were homogenised in ChABC buffer (40 mM Tris–HCl, pH 8.0, 40 mM sodium acetate) containing Benzonase and Halt protease/phosphatase inhibitors (Thermo Scientific) and pelleted, and the supernatant containing soluble protein fraction was separated from the pellet (insoluble fraction), which was resuspended in ChABC buffer. One aliquot of each fraction was incubated with ChABC for 12 h at 37°C, then heated for 5 min at 95°C, separated by SDS–PAGE and transferred onto PVDF membranes. Blocking was for 1 h with 5% milk in TBST overnight at 4°C with primary antibodies (1:100, anti‐CS‐0S, 1B5; anti‐CS‐4S, 2B6, antiCs‐6S, 3B3; Amsbio). Blots were washed 3 × 5 min in TBST and incubated with HRP‐conjugated secondary anti‐mouse‐IgG‐H&L chain or anti‐rabbit‐IgG‐F(ab')2 (GE Healthcare) antibody for 1 h at RT, washed 3 × 5 min in TBST and visualised.
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