Q: Why is the activity of my target protein low?
A: Two common reasons are:
- Incorrect folding. Try reducing the reaction temperature to promote proper folding.
- Missing post-translational modifications (PTMs). This kit is a prokaryotic system and does not provide eukaryotic PTMs. Ensure your protein’s activity does not depend on PTMs, or consider using a system that supplies them.
Q: The positive control expresses, but my target shows low or no expression. What should I check?
A: Verify the following, in order:
1. DNA contamination
- Symptoms/Reason: Template contaminated with residual ethanol or RNases.
- Solutions:
- Prepare a fresh DNA template and ensure all ethanol is fully removed after precipitation.
- Use strict RNase-free technique (gloves, masks, nuclease-free plastics/reagents).
2. Insufficient DNA template
- Reason: Template amount is below the effective range.
- Solution: Use a final DNA concentration of 10–40 ng/µL in the reaction (typical working window).
3. Template design or tag issues
- Reasons: Misplaced ATG start codon, reading-frame errors, suboptimal codon usage for E. coli, or non-ideal tag configuration.
- Solutions:
- Ensure the start codon is correctly positioned for expression and the reading frame is intact.
- Codon-optimize the coding sequence for a prokaryotic (E. coli) system.
- If applicable, verify tag choice and orientation (N- vs C-terminal) and re-design if needed.
Q: How do I decide whether my gene needs redesign or further optimization?
A: If the positive control performs well but your target does not, prioritize:
(i) fresh, clean DNA;
(ii) template amount;
(iii) start codon/frame;
(iv) codon optimization and tag configuration.
Adjust one variable at a time to identify the limiting factor.
Q: Could my fusion tag be hurting expression?
A: Yes. Tag type and position (N- vs C-terminal) can alter mRNA structure or translation efficiency.
Solutions:
- Try moving the tag (N ↔ C terminus).
- Switch tag type (e.g., His₆ ↔ MBP/GST) and/or add a flexible linker (e.g., G₄S repeats).
- Re-check the 5′ RBS–AUG context and avoid strong RNA structures near the start codon.
Q: My target protein is very large and yield is poor—what can I do?
A: Yield typically drops as molecular weight increases.
Solutions:
- Lower reaction temperature (e.g., within your kit’s recommended range) and extend incubation time.
- If supported by your kit, supplement amino acids and ATP/energy mix.
- Consider domain constructs (N-/C-terminal domains) or optimize tags for solubility.
- Scale up volume and ensure consistent mixing.
Q: The protein aggregates or precipitates—how can I improve solubility?
A: Aggregation often indicates expression outpaces folding or the protein is hydrophobic.
Solutions:
- Reduce temperature to slow synthesis and promote folding.
- Lower DNA amount to moderate expression rate.
- For hydrophobic targets, add a mild, compatible non-ionic detergent (e.g., Tween-20 at 0.01–0.05%) to the reaction.
- Evaluate alternative tags (solubility enhancers) and/or add glycerol to the analysis buffer.
- After expression, spin down and analyze supernatant (soluble) vs pellet fractions.
Q: I see multiple, unexpected bands on SDS-PAGE—what’s going on?
A: Two common causes are sample denaturation issues and internal translation starts.
(a) Insufficient SDS in sample buffer
Solutions:
- Prepare fresh SDS-PAGE sample buffer (ensure correct SDS and reducing agent).
- Heat denature (e.g., 95 °C, 3–5 min) before loading.
(b) Internal initiation from a cryptic RBS + internal ATG
Reason: A Shine–Dalgarno–like sequence near an internal ATG (Met) can trigger unintended starts.
Solutions:
- Scan the coding sequence for RBS-like sites appropriately spaced (~5–10 nt) upstream of internal ATGs.
- Disrupt the cryptic RBS with synonymous mutations (codon optimization).
- If functionally permissible, mutate the internal ATG (Met) to a non-start codon (synonymous/ conservative change) to prevent re-initiation.
- Strengthen the intended 5′ RBS–start context to bias initiation at the correct AUG.
Q: How do I prevent spurious bands caused by unintended internal translation starts?
A: Internal Shine–Dalgarno–like sequences upstream of an internal ATG can trigger alternative starts.
Solutions:
- Introduce synonymous point mutations to disrupt the cryptic RBS or alter its spacing.
- Change the RBS sequence near the internal ATG to reduce unintended initiation.
- If functionally acceptable, mutate the internal ATG (Met) to a non-start codon.
Q: I see “tailing”/smearing on my polyacrylamide gel—what’s causing it?
A: Common causes and fixes:
- Excessive protein loading → Reduce the amount loaded.
- Gel not clean/fresh → Pour a new gel; rinse the gel briefly with running buffer before loading.
- Ethanol carryover from DNA purification → Ensure residual ethanol is fully removed during DNA prep.
- Expired precast gel → Do not use expired gels; switch to a fresh lot.
Q: My positive control shows no fluorescence—what should I check first?
A: Two frequent issues are reagent integrity and RNase contamination.
Solutions:
- Reagents inactivated → Store all kit reagents at −80 °C. For diluted mixes, aliquot and snap-freeze in liquid nitrogen, then store at −80 °C to avoid repeated freeze–thaw.
- RNase contamination → Wear gloves and a mask; use RNase-free tips, tubes, and reagents throughout setup.