Hippocampal tissues and cultured neurons were lysed in buffer containing 25 mM HEPES at pH7.9, 150 mM NaCl, 1 mM PMSF, 20 mM NaF, 1 mM DTT, 0.1% NP40, and proteinase inhibitor cocktails (Roche). Protein concentrations were determined by Folin phenol method with bovine serum albumin as standard. Twenty micrograms of the protein was separated on 8–12% SDS-PAGE gels (Bio-Rad) and transferred to PVDF membranes (Millipore). The membranes were blocked in 5% BSA in TBS-T with 0.05% Tween-20 and incubated with primary antibodies at 4°C overnight. Dilutions of primary antibodies were 1:1000 for UTX (E409, Millipore), H3K27me3 (1:1,000, 07449, Millipore), PSD95 (ab2723, Abcam), Synapsin (ab8049, Abcam), and 1:10,000 for β-actin antibody (Sigma). As for the secondary antibodies, we used HRP-linked goat anti-mouse or HRP-Linked goat anti-rabbit at 1:500. Enhanced chemoluminescence (ECL, Pierce) was used for detection. Quantification of the blots was determined with Quantity One Ver.4.4.0 (BioRad, USA).
Quantity one ver 4
Manufactured by Bio-Rad
Sourced in United States
Quantity One Ver.4.4.0 is a software application developed by Bio-Rad for quantitative analysis of gel and blot images. The software provides tools for image capture, analysis, and data management.
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2 protocols using quantity one ver 4
1
Characterization of Hippocampal Proteins
2
Western Blot Analysis of 5-HT5B Receptor
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Cultured neurons were lysed in buffer containing 25 mM HEPES at pH7.9, 150 mM NaCl, 1 mM PMSF, 20 mM NaF, 1 mM DTT, 0.1 % NP40, and proteinase inhibitor cocktails (Roche). 10 μg of the protein was separated on 8–12 % SDS-PAGE gels (Bio-Rad) and transferred to PVDF membranes (Millipore). The membranes were blocked in 5% BSA in TBS-T with 0.05 % Tween-20 and incubated with primary antibodies at 4 °C overnight. Dilutions of primary antibodies were 1:1000 for 5-ht5b (TA315779, OriGene) and 1:10000 for β-actin antibody (Sigma). HRP-linked goat anti-mouse or HRP-Linked goat anti-rabbit were used as secondary antibodies. Dilutions of secondary antibodies were 1:1000. Enhanced chemoluminescence (ECL, Pierce) was used for detection. Quantification of the blots was determined with the use of a Quantity One Ver.4.4.0 (BioRad, USA).
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