Cell Surface Biotinylation of Mouse Brain Slices

Cell surface biotinylation of mouse brain slices was conducted as published [62 (link)]. After euthanization, mouse brains were freshly isolated and sliced into 1 mm coronal sections. Brain slices were incubated in 1 mg/ml sulfo-NHS-SS-biotin (ThermoFisher) in pre-chilled oxygenated aCSF on ice for 30 min and then washed twice with 50 mM glycine and three times with 1 mg/ml BSA in pre-chilled oxygenated aCSF. Hippocampus was dissected from slices, extracted and the biotinylated proteins purified using NeutrAvidin beads (ThermoFisher) and processed for Western blot with following antibodies: rabbit anti CLC-3 (Rabbit, 1:1000, Alomone, ACL-001), Rabbit anti-Na-K ATPase (1:10,000, Abcam, ab76020). Blots were developed with goat anti-mouse IRDye 680RD and goat anti-rabbit IRDye 800CW (1:5000, LI-COR) and Odyssey CLx imager (LI-COR) and quantitated using Image Studio (LI-COR).

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Wu Z., Huo Q., Ren L., Dong F., Feng M., Wang Y., Bai Y., Lüscher B., Li S.T., Wang G.L., Long C., Wang Y., Wu G, & Chen G. (2019). Gluconate suppresses seizure activity in developing brains by inhibiting CLC-3 chloride channels. Molecular Brain, 12, 50.

Publication 2019

sulfo-NHS-SS-biotin NeutrAvidin beads Rabbit anti-Na-K ATPase goat anti-mouse IRDye 680RD goat anti-rabbit IRDye 800CW Odyssey CLx imager Image Studio

Antibodies Biotinylation Brain Cell Glycine Goat Hippocampus Irdye 800cw Mouse Na k atpase Neutravidin Proteins Rabbit Sulfo nhs biotin Western blot

Corresponding Organization :

Other organizations : Pennsylvania State University, South China Normal University, Fudan University, Shanghai Jiao Tong University, Sun Yat-sen University

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Variable analysis

independent variables

Exposure of mouse brain slices to 1 mg/ml sulfo-NHS-SS-biotin in pre-chilled oxygenated aCSF on ice for 30 min

dependent variables

Abundance of biotinylated proteins in the hippocampus

control variables

Mouse brain slices
Coronal sections of 1 mm thickness
Washing of brain slices with 50 mM glycine and 1 mg/ml BSA in pre-chilled oxygenated aCSF
Dissection of hippocampus from brain slices
Purification of biotinylated proteins using NeutrAvidin beads
Western blot analysis with antibodies against CLC-3 and Na-K ATPase
Imaging and quantification using Odyssey CLx imager and Image Studio software

positive controls

Not specified

negative controls

Not specified

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