TRPC5-tranfected 293T cells were incubated with Sulfo-NHS-Biotin [0.5 mg/ml in phosphate-buffered saline (PBS), Aladdin] for 30 min and washed thrice with 50 mM glycine in PBS. Cells were lysed and aliquoted for extraction of plasma membrane proteins and total proteins separately. Biotinylated plasma membrane proteins were captured with streptavidin magnetic beads (Invitrogen). Western blotting was then performed to detect TRPC5 in the plasma membrane and total proteins. The ratio between biotinylated and total TRPC5 proteins was used to represent the level of TRPC5 proteins expressed at the cell surface. TRPC4 constructs were analyzed in the same way. Primary antibodies against TRPC5 and TRPC4 were purchased from Sangon Biotech (Shanghai, China) and Proteintech (Wuhan, China), respectively.

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

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.