TY  - JOUR
TI  - Biotinylation and Purification of Plasma Membrane-associated Proteins from Rodent Cultured Neurons
AU  - Caldeira, Margarida 
AU  - Ferreira, Joana S.
AU  - Carvalho, Ana Luísa
AU  - Duarte, Carlos B.
VL  - 6
IS  - 10
PY  - 2016
DA  - 2016/05/20
SP  - e1807
C1  - Bio-protocol 2016;6:e1807
DO  - 10.21769/BioProtoc.1807
UR  - https://doi.org/10.21769/BioProtoc.1807
AB  - This protocol aims at the biotin labeling and affinity purification of plasma membrane proteins from cultured neurons. Protein biotinylation consists in the covalent attachment of biotin to proteins. Biotin is a membrane unpermeable molecule with a small size (MW 244.31 g/mol) and therefore does not interfere with the normal function of proteins. Biotin binds to streptavidin and avidin molecules with high affinity. This binding is extremely resistant to temperature, pH and proteolysis, which allows capture and purification of plasma membrane proteins. Moreover, proteins can bind several biotin molecules, that will allow the consequent binding of several streptavidin or avidin molecules, increasing the sensitivity of detection of the proteins of interest. In this protocol proteins at the cell surface of live cultured neurons are biotinylated. Neuronal extracts are prepared and biotinylated proteins are collected with NeutrAvidin-coupled beads, and analyzed by Western blotting.
KW  - Plasma-membrane proteins
KW  - Affinity purification
KW  - Biotin
KW  - Cell surface
KW  - Cultured neurons
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False