Western Blot Protocol

Western Blot Protocol

Comprehensive guide for Western Blotting using LabReCon antibodies.

Materials

• Protein samples
• SDS-PAGE gel and running buffer
• Transfer membrane (PVDF or nitrocellulose)
• Blocking buffer (5% milk or BSA in TBST)
• Primary antibody (LabReCon)
• HRP-conjugated secondary antibody
• ECL substrate

Procedure

1. Prepare protein samples in Laemmli buffer and boil for 5 min
2. Load 20-40 μg protein per lane on SDS-PAGE gel
3. Run at 120V until dye front reaches the bottom
4. Transfer to membrane at 100V for 1 hour (wet transfer)
5. Block membrane in 5% milk/TBST for 1 hour at RT
6. Incubate with primary antibody (1:1000) overnight at 4°C
7. Wash 3×10 min with TBST
8. Incubate with secondary antibody (1:5000) for 1 hour at RT
9. Wash 3×10 min with TBST
10. Develop with ECL substrate and image