Biochemical measurement of surface-expressed receptors
This protocol is extracted from research article:
GPR40 modulates epileptic seizure and NMDA receptor function
Sci Adv, Oct 17, 2018; DOI: 10.1126/sciadv.aau2357

Biochemical measurement of surface-expressed receptors was performed as previously described (44). Briefly, brain tissues were washed in wash solution and homogenized in permeabilization buffer. According to the instructions for the Mem-PER Plus Membrane Protein Extraction Kit for Soft Tissue (Thermo Fisher Scientific, Waltham, MA), the homogenate was incubated at 4°C for 10 min and centrifuged at 16,000g at 4°C for 15 min, and supernatant fractions were collected. A sample of 15 μg of protein from the supernatant was used to determine total protein expression. To determine surface expression, 150 μg of protein from the supernatant was incubated with 100 μl of 50% NeutrAvidin agarose (Pierce Chemical Co.) at 4°C overnight, and the bound proteins were resuspended in SDS sample buffer and boiled for 10 min. Western blotting was performed to detect both total and surface protein expression.

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