Surface protein biotinylation
Pietro De Camilli, Daehun Park
Abstract
This protocol describes surface protein labeling with biotin using EZ-Link Sulfo-NHSLC-Biotin. This chemical reacts with primary amines such as lysine but does not permeate cell membranes because of the charge. Thus, it only biotinylates surface proteins.
Attachments
Steps
Surface protein biotinylation
Wash cells with ice-cold PBS.
Wash cells with ice-cold PBS. (1/3)
Wash cells with ice-cold PBS. (2/3)
Wash cells with ice-cold PBS. (3/3)
Incubate cells with EZ-Link Sulfo-NHS-Biotin for 0h 30m 0s at 4°C to label surface proteins.
Discard biotin containing medium. Quench and remove unbound biotin using 50millimolar (mM) glycine in ice-cold PBS for 0h 10m 0s at4°C.
Wash 2-3 times with ice-cold PBS.
Wash with ice-cold PBS. (1/3)
Wash with ice-cold PBS. (2/3)
Wash with ice-cold PBS. (3/3)
Lysis cells with 1% triton X-100 lysis buffer and centrifuge the samples at 14000x g,4°C.
Collect supernatants and discard the pellets.
Measure the protein concentrations using BCA Protein Assay Kit.
Incubate the same amount of lysates (500µg to 1000µg) with streptavidin or NeutraAvidin beads for 2h 0m 0s to 2h 0m 0s at4°C to pull-down the biotinylated proteins.
Wash the beads with lysis buffer by cycles of suspension.
Wash the beads with lysis buffer by cycles of suspension. (1/3)
Wash the beads with lysis buffer by cycles of suspension. (2/3)
Wash the beads with lysis buffer by cycles of suspension. (3/3)
Centrifugation and elute proteins from the packed beads by adding an equal volume of 2x sample buffer and boiling for 0h 5m 0s 95°C.
Run eluate samples on a SDS polyacrylamide gel and perform western blotting to visualize labeling (and thus evidence of surface expression) of the protein of interest. Total cell lysates can be used to determine the expression level.
