Brain samples were homogenized in Tissue-Protein Extraction Reagent buffer (ThermoFisher Scientific, #78510; Rockford, IL) supplemented with commercially available protease and phosphatase inhibitor cocktail tablets (Roche, #04693116001, #049068445001; Indianapolis, IN), and using an ultrasonic homogenizer. Homogenates were cleared by centrifugation at 14,000 rpm for 10 min at 4°C, and the supernatant was collected as total homogenate and stored at −80°C until use. Protein concentration in the samples was determined with bicinchoninic acid method (ThermoFisher Scientific, #23225; Rockford, IL), using bovine serum albumin as standard.
Tissue protein extraction reagent buffer
Manufactured by Thermo Fisher Scientific
Tissue-Protein Extraction Reagent buffer is a reagent designed for the extraction of proteins from tissue samples. It provides a consistent and efficient way to isolate proteins for further analysis.
Automatically generated - may contain errors
2 protocols using tissue protein extraction reagent buffer
1
Brain Homogenization and Protein Quantification
2
Protein Extraction and Western Blot
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Samples were processed and homogenised as described in the section on RNA isolation. Proteins were extracted using a tissue protein extraction reagent buffer (Thermo Fisher Scientific) supplemented with proteinase and phosphatase inhibitors (Roche). Protein content of 3D-LTCs was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). For blotting, nitrocellulose membranes were used. After blocking with 5 wt-% nonfat dry milk (AppliChem) for 1 h, blots were incubated with primary antibodies at 4°C overnight or for 1.5 h at room temperature. Secondary antibodies were incubated for 1.5 h. For detection, an enhanced chemoluminescence substrate (Thermo Fisher Scientific) was used and blots were imaged using a ChemiDoc XRS+ (BioRad, Hercules, CA, USA). The antibodies used are listed in online supplementary table S4.
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