📋 Workflow
Full workflow, products add-to-cart directly1
Lysis and clarification
1 hLyse by sonication/high-pressure, centrifuge supernatant and filter to remove particulates
💡 Tip Add protease inhibitors to prevent degradation
⚠️ Caution Keep cold throughout
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2
Affinity capture
2 hLoad onto affinity column/beads, wash off contaminants, elute with imidazole or competitor
💡 Tip Titrate imidazole to separate binding/elution
⚠️ Caution Load within column capacity
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3
Fine purification (SEC/IEX)
2–3 hUse ion exchange or size-exclusion chromatography to remove remaining impurities and exchange buffer
💡 Tip SEC also performs buffer exchange
⚠️ Caution Balance and calibrate SEC column
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4
Purity and concentration check
1 hAssess purity by SDS-PAGE, measure concentration by BCA or A280, verify by MS/activity if needed
💡 Tip Pool high-purity fractions and concentrate
⚠️ Caution Concentration may cause aggregation/precipitation
🧪 Materials for this step (click to shop by spec/brand)
⚠️ 5 Common Beginner Mistakes
- ① Protein degrades during purification
- ② Wrong imidazole loses protein or lowers purity
- ③ Concentration causes aggregation/precipitation
- ④ Incomplete lysis lowers recovery
- ⑤ Buffer mismatch reduces solubility
❓ FAQ
+How to judge sufficient purity?
Single clean band/peak in SEC, typically >95% for applications.
+Aggregation during purification?
Add reductant, solubilizer, adjust pH/salt, or stabilizing buffers.
+Can one affinity step reach high purity?
Often combined; affinity + SEC/IEX yields high purity.