Western blot analysis

PV Preeti Dinesh Virwani
LC Lin Cai
PY Patrick Ka Kit Yeung
GQ Gordon Qian
YC Yingxian Chen
LZ Lei Zhou
JW Jason Wing Hon Wong
YW Yu Wang
JH Joshua Wing Kei Ho
KL Kui Kai Lau
PQ Pei-Yuan Qian
SC Sookja Kim Chung
request Request a Protocol
ask Ask a question
Favorite

Radioimmunoprecipitation assay (RIPA) buffer was used to extract proteins from EWAT and jejunum. Protein concentration in the lysate was measured using a protein dye reagent (Bio-Rad, Cat. No. 500-0006, USA) as per the manufacturer’s instructions. The proteins were separated by 10% of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein samples (25μg) and the protein marker were loaded in the wells and separated by SDS-PAGE at 70V for 3–4 h. The transfer was allowed to occur overnight in a cold room maintained at 4 °C, under 30V. The membranes were blocked with 5% of nonfat milk (Bio-Rad, Cat. #170-6404) in TBS-T (Tris-HCl, 50mM; NaCl, 150mM; Tween20, 0.1%), and immunoblotting was carried out using Epac1 (Cell Signaling #4155) and GAPDH (Abcam #9484) antibodies.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A