Protein Analysis
Standard Western Blotting for Protein Detection (Mock Protocol)
DBC
Dr. Ben Carter
Center for Genomic Research
Aim / Objective
A standard protocol for separating proteins by size via gel electrophoresis, transferring them to a membrane, and identifying a specific protein using antibodies. From protocols.io.
Protocol Steps
1. Prepare protein samples in Laemmli buffer and boil for 5 mins.
2. Load samples onto a 4-20% SDS-PAGE gel and run at 120V for 90 mins.
3. Transfer proteins to a PVDF membrane using a wet transfer system at 100V for 1 hour.
4. Block the membrane with 5% non-fat milk in TBST for 1 hour at room temperature.
5. Incubate with primary antibody (e.g., 1:1000 dilution) overnight at 4°C.
6. Wash membrane 3x for 10 mins with TBST.
7. Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour at RT.
8. Wash membrane 3x for 10 mins with TBST.
9. Add ECL chemiluminescent substrate and image the blot using a digital imager.
Submit Your Review
You must be logged in to contribute.
Community Reviews (0)
No contributions yet. Be the first to add one!
Actions
Information
Keywords
Western Blot
Immunoblotting
Protein Detection
SDS-PAGE
Rating
4.5 / 5
Likes230
PublishedMarch 20th, 2026
Reviews45