When preparing to run SDS-PAGE, selecting the correct acrylamide gel percentage is one of the most critical decisions. It directly affects the resolution, separation range, and downstream applications such as Western blotting. This guide will walk you through how to choose the right precast gel % based on:
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Protein molecular weight
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Separation goals (e.g. band resolution vs. range)
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Buffer systems
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Transfer compatibility
🔍 What Does Gel Percentage Actually Mean?
Acrylamide gel concentration (e.g. 10%, 12%, 4–20%) determines pore size in the gel matrix. This affects how easily proteins migrate based on size:
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Low % gels have larger pores, allowing large proteins to move efficiently.
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High % gels have smaller pores, making them ideal for resolving small proteins or closely sized bands.
🧠 Choosing Gel % by Molecular Weight (with Common Targets)
| Acrylamide % | Best for MW Range | Example Proteins |
|---|---|---|
| 6% | > 200 kDa | Spectrin, Titin, large IgG complexes |
| 8% | 100–200 kDa | Fibrinogen, β-galactosidase |
| 10% | 60–150 kDa | BSA, GAPDH, actin, HSP70 |
| 12% | 20–100 kDa | Histones, caspases, transcription factors |
| 15% | < 30 kDa | Small peptides, cytokines, ubiquitin |
| 4–20% | 10–200+ kDa | Multi-target analysis, unknown proteins |
🧪 Tip: If you're unsure about your protein size or analyzing multiple targets, start with a 4–20% gradient gel.
📦 Precast Gel Formats & Tank Compatibility
NuSep offers precast gels in three formats to match your tank system:
| Model | Tank Compatibility | Size |
|---|---|---|
| NB | Bio-Rad® Mini-PROTEAN® | 10 x 8 cm |
| NG | Hoefer® / Cytiva™ | 10 x 10 cm |
| NN | Invitrogen™ Novex® | 8 x 8 cm |
👉 Visit our Precast Gels Collection to explore models by tank type and %.
🔬 Resolution Goals vs. Gel %
When you have closely sized protein targets, a higher % gel will give better band separation.
When you have proteins spanning a wide MW range, use a gradient gel.
| Use Case | Recommended Gel % |
|---|---|
| Two proteins at 40 kDa and 45 kDa | 12–15% |
| Multiple unknown sizes | 4–20% |
| Large protein detection only | 6–8% |
⚠️ Transfer Compatibility Tips for Western Blotting
Higher % gels can slow the transfer of large proteins during blotting due to tight pore size. Here’s how to optimize:
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For proteins >150 kDa, use 6–8% gels and wet transfer (overnight)
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For small proteins <30 kDa, use 12–15% gels and PVDF membranes with shorter transfer time
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Consider pre-stained MW markers in the same % gel to track mobility
🔄 Buffer System Considerations
Most labs use Tris-Glycine-SDS running buffers. Ensure your buffer is matched with the gel format:
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NuSep precast gels are optimized for standard TGS buffers
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Use our SingleShot Buffers for error-free prep and reproducibility
⚗️ Tip: Even minor variations in homemade buffers (e.g. pH, ionic strength) can skew migration patterns. Use pre-formulated buffers to avoid inconsistency.
🔧 Additional Tips
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Don't overload lanes — higher % gels are less forgiving with overloading and can distort bands
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Pre-run the gel 5 minutes before loading to stabilize the electric field
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If bands are smearing, check:
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Sample prep (e.g. boil time, reducing agent)
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Over-concentration
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Incorrect buffer pH or salt
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🧪 Final Takeaway:
Choosing the right gel % isn’t just about MW — it’s about your goal: resolution, range, and reproducibility. Use gradient gels for flexibility and higher % gels for precision.
✅ Explore our full range of NB, NG, and NN model gels here
📩 Need help choosing? Email our team