📋 Workflow
Full workflow, products add-to-cart directly1
Construct expression vector
1–2 daysClone target gene into tagged (His/GST) expression vector with promoter and selection marker
💡 Tip Tag facilitates downstream purification/detection
⚠️ Caution Choose promoter compatible with host
🧪 Materials for this step (click to shop by spec/brand)
2
Transform and seed culture
1 dayTransform competent expression host, streak single colonies, inoculate antibiotic medium
💡 Tip Pick single colonies for uniformity
⚠️ Caution Maintain antibiotic selection for plasmid
🧪 Materials for this step (click to shop by spec/brand)
3
Induction
4–6 hGrow to suitable OD600, add IPTG, induce at lower temp for hours to favor soluble expression
💡 Tip Low temp slow induction improves solubility
⚠️ Caution Optimize induction temperature and IPTG concentration
🧪 Materials for this step (click to shop by spec/brand)
4
Harvest and detection
1 hHarvest cells by centrifugation, lyse, take soluble/supernatant and pellet, check by SDS-PAGE/WB
💡 Tip Check both soluble and insoluble fractions
⚠️ Caution Include uninduced control for comparison
🧪 Materials for this step (click to shop by spec/brand)
⚠️ 5 Common Beginner Mistakes
- ① Low or abnormal expression
- ② Protein forms insoluble inclusion bodies
- ③ Poor induction causes degradation or toxicity
- ④ No induction control confounds expression assessment
- ⑤ Vector/sequence errors prevent expression
❓ FAQ
+Why protein mostly in pellet?
Common for fast/complex proteins; lower temp, slow induction or solubility tags help.
+How to increase yield?
Optimize promoter, codon usage, host strain, induction timing and culture conditions.
+Can E. coli express all proteins?
No, complex/modified or glycosylated proteins need eukaryotic hosts like yeast or mammalian cells.