immunoblotting (JMS protocol)
Veerle Baekelandt
Published: 2021-08-05 DOI: 10.17504/protocols.io.bthcnj2w
Abstract
imonoblotting protocol JMS
Steps
1.
determination of protein content by a bicinchoninic acid (BCA) assay
2.
cell extracts containing 15 µg of total protein were separated on 4-15% sodium dodecyl sulfate-polyacrylamide gradient (150V, Bio-Rad) gels
3.
electroblot gel onto polyvinylidene difluoride membrane
4.
incubate membranes with 0.4% volume paraformaldehyde for 0h 15m 0s for better protein binding
5.
Block membranes with 5Mass Percent milk for 0h 30m 0s
6.
incubate blot with primary antibody for 4h 0m 0s
7.
wash blot for 3 times for 0h 5m 0s with PBS-0.1% volume TRITON-X100
8.
incubate for 1h 0m 0s with horseradish peroxidase-conjugated secondary antibody (Dako, Glostrup)
9.
detect using chemiluminescence
