The prepared membrane homogenates (50 μl) were plated in triplicate in poly-d-lysine–coated plates and spun at 2500g for 10 min at 4°C. One hundred microliters of a solution of 2% (w/v) glutaraldehyde in PBS was added and incubated 15 min at room temperature. Supernatant was removed, and 100 μl of 0.1 M glycine and 0.1% BSA in PBS was added to each well for 30 min at room temperature. Liquid was aspirated and wells were blocked in 5% (w/v) milk in PBS for 30 min at room temperature. Anti-HA antibody (1:2000) in 5% (w/v) milk was added to wells and incubated for 1.5 hours at room temperature. Wells were washed three times in PBS before adding secondary antibody (anti-mouse HRP, 1:5000) in 5% (w/v) milk for 45 min at room temperature. Wells were washed three times in PBS, and 50 μl of substrate (3,3′,5,5′-tetramethylbenzidine; Sigma, T0440) was added for 5 min at room temperature. Reactions were terminated by adding 50 μl of 10% (v/v) sulfuric acid, and absorbance at 450 nm was measured on a SpectraMax i3 plate reader (Molecular Devices). Results from GTPγS binding assays were normalized to GHSR1a levels for each sample.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.