📋 Protocol
Full flow at a glance, add products directly1
Protein Extraction (cell lysis)
30 minLyse cells with lysis buffer containing protease inhibitors; centrifuge and collect the supernatant (protein solution).
💡 Tip Keep everything on ice to prevent protein degradation.
⚠️ Caution Add protease/phosphatase inhibitors before lysis, or you'll lose your phospho-proteins.
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2
Protein Quantification (BCA)
30 minUse a BCA kit to measure protein concentration so every lane is loaded equally.
💡 Tip Run a BSA standard curve in the same plate for accurate quantification.
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3
SDS-PAGE Electrophoresis
1.5 hDenature proteins, load samples, and separate them by molecular weight in the gel.
💡 Tip Denature samples at 95°C for 5 min with loading buffer before loading.
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4
Transfer (wet / semi-dry)
1 hTransfer proteins from the gel onto a PVDF or NC membrane.
⚠️ Caution PVDF membrane must be pre-activated in methanol (~15 s) before equilibration in transfer buffer.
⚠️ Caution Gel faces the cathode (−), membrane faces the anode (+), so proteins migrate toward the membrane.
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5
Blocking
1 hBlock non-specific binding sites on the membrane with non-fat milk or BSA.
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6
Antibody Incubation (primary + secondary)
过夜 / 1 hFirst incubate with a primary antibody against your target, then with a labeled secondary antibody that binds the primary.
💡 Tip Wash 3×5–10 min with TBST after each antibody incubation to reduce background.
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7
Detection (ECL Chemiluminescence)
15 minECL substrate reacts with HRP to emit light; capture with an imaging system.
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⚠️ 5 Common Beginner Mistakes (Pitfall Guide)
- ① Transfer direction reversed: gel on cathode (−), membrane on anode (+), or proteins run into buffer and the experiment fails.
- ② Primary antibody too concentrated: causes a black background. Dilute per datasheet first, then increase slowly.
- ③ Unequal loading: always quantify protein first, or band intensity is meaningless.
- ④ PVDF membrane not methanol-activated: the hydrophobic membrane won't bind protein.
- ⑤ Insufficient washing: wash 3× with TBST after blocking and antibody steps, or background stays high.
❓ FAQ (Troubleshooting)
+What if my bands are smeared?
Usually caused by sample degradation, overloading, or high voltage. Keep samples on ice, reduce loading amount, and lower the voltage.
+Why is my transfer incomplete (high MW proteins missing)?
Extend transfer time, add SDS to transfer buffer, or reduce methanol for high-MW proteins.
+No signal at all — what went wrong?
Check: (1) primary/secondary antibody mismatch, (2) forgot ECL substrate, (3) transfer direction, (4) antibody too diluted.