When you’re prepping for SDS-PAGE, selecting the right gel is more than just grabbing whatever’s available — it directly affects how well your proteins separate and whether you can even see your target bands. This article breaks down the core differences between fixed % acrylamide gels and 4–20% gradient gels, so you can confidently choose the best format for your experiment. Whether you're analyzing large complexes, small peptides, or multiple targets in one run, the right gel will save time and give you sharper, more reproducible results.
Fixed % Gels
-
Made with one acrylamide concentration throughout (e.g. 10%, 12%, or 15%)
-
Pore size is uniform across the gel
-
Best for resolving proteins within a narrow molecular weight range
Gradient Gels (e.g. 4–20%)
-
Acrylamide concentration increases gradually from the top (stacking zone) to the bottom (resolving zone)
-
Pore size narrows from top to bottom
-
Enables separation of a wide range of protein sizes in a single run
When to Use Fixed % Gels
Use fixed % gels when:
-
You know your target protein's approximate size
-
You want maximum resolution around a specific molecular weight
-
You're running similar-sized proteins across multiple lanes
Examples:
Gel % | Protein Range | Best For |
---|---|---|
10% | 50–150 kDa | Actin, tubulin, HSP70 |
12% | 20–100 kDa | Enzymes, transcription factors |
15% | <30 kDa |
Ubiquitin, small peptides |
🎯 Tip: Fixed % gels are ideal for labs that focus on 1–2 known targets and require sharp, clean bands in that range.
🧪 When to Use 4–20% Gradient Gels
Use gradient gels when:
-
Your protein mixture contains multiple sizes
-
You're working with unknown molecular weights
-
You want to visualize full molecular weight ladders
-
You're analyzing post-translational modifications or protein degradation products
Why Gradient Gels Are Versatile:
-
Small proteins resolve at the top (low acrylamide %)
-
Large proteins resolve at the bottom (high acrylamide %)
-
This allows high and low MW bands to separate cleanly on one gel
🧪 Use case: Western blots where you want to probe both a phosphorylated 25 kDa target and its full-length 80 kDa form — a gradient gel is perfect.
⚖️ Comparison Table: Gradient vs. Fixed % Gels
Feature | Fixed % Gel | 4–20% Gradient Gel |
---|---|---|
Pore Size | Uniform | Varies (top to bottom) |
Resolution Range | Narrow | Wide |
Best For | Known MW proteins | Mixed/unknown sizes |
Run Time | Slightly faster | Slightly longer |
Price | Lower | Slightly higher |
Reproducibility | High | High (precast) |
Visualization | Sharp bands at specific MW | Ladder-like separation across range |
🧪 NuSep’s Precast Gel Options
NuSep offers a wide range of fixed % gels (10%, 12%, 15%) and 8-16%, 4–20% gradient gels across three compatibility formats:
-
NB Series – Bio-Rad® Mini-PROTEAN® tanks
-
NG Series – Cytiva™ / Hoefer® systems
-
NN Series – Invitrogen™ Novex® tanks
✅ All gels are leak-resistant, shelf-stable, and come with tight well formation for clean, reproducible results.
❓FAQs
Q: Do gradient gels require a special tank?
A: No — as long as the gel cassette fits your tank (e.g. Mini-Pro, Bio-Rad, Hoefer, Invitrogen), gradient and fixed % gels run the same way.
Q: Can I cut gradient gels for transfer?
A: Yes, just be mindful that protein migration rates vary across the gel — always use molecular weight markers.
Q: Do gradient gels affect blotting efficiency?
A: Slightly — very large proteins may transfer more slowly from dense (high %) zones. Use extended transfer times if needed.