Western Blotting Protocol

Overview of all solutions, buffers, and reagents required for the protocol — RIPA lysis buffer, Laemmli sample buffer, running buffer, transfer buffer, blocking solution, TBST wash buffer, primary and secondary antibodies, ECL substrate.

Lyse cells, quantify protein, mix with Laemmli buffer and denature. Load samples and ladder onto SDS-PAGE gel, run electrophoresis, then transfer proteins onto a PVDF or nitrocellulose membrane.

Block the membrane with 5% non-fat dry milk or BSA in TBST to prevent non-specific binding. Incubate with primary antibody overnight at 4 C, wash with TBST, then incubate with HRP-conjugated secondary antibody.

Wash the membrane with TBST to remove unbound secondary antibody, then apply ECL chemiluminescent substrate. Capture the signal on film or a digital imaging system.

Key reminders on why each step matters: proper sample lysis ensures complete protein extraction, correct gel percentage matches target MW, transfer time and voltage affect membrane signal, and antibody dilutions per datasheet directly impact band quality. Skipping or modifying steps often causes the most common Western blot failures.