Jul 28, 2023
Cell surface biotinylation
- 1KU Leuven
Protocol Citation: rosanne.wouters, Peter Vangheluwe 2023. Cell surface biotinylation. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9d9o4g3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 01, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82746
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Abstract
The protocol describes cell surface biotinylation to identify plasma membrane localized proteins in cell culture via western blotting.
grown cells until 70-80% confluency in 10cm dish
place cells on ice and wash with ice-cold PBS
incubate cells for 30 min c on ice with PBS containing 2.5 mg/ml Sulfo-NHS-SS-biotin (Pierce).
stop the biotinylation reaction by washing 3 times for 5min with quenching solution (0.5% BSA and 100 mM glycine in PBS).
collect cells by scraping in PBS and centrifugation 500 rpm, 4°C, 00:05:00
5m
lyse cells by resuspending cell pellet RIPA buffer supplemented with protease inhibitor and incubating for 30min on ice
clear cell lysate by centrifugation (20min, 14000 gavg, 4°C) and collect supernatant
isolate biotinylated proteins with immobilized Neutravidin beads
wash neutravidin beads 2 times with RIPA buffer
add supernatant from step 7 to beads
incubate while rotating head-over-head 02:00:00 at 4°
2h
wash neutravidin beads 3 times with RIPA buffer
Input and bound proteins were processed for Western blotting with 4x LDS loading buffer.
remove all RIPA buffer from neutravidin beads
add 4x LDS loading buffer
denature proteins for 10min, 70°C
proceed with Western blotting