Gel vs. Gel: Stacking vs. Separating Explained
A “stacking gel” is the loose top layer in SDS-PAGE that aligns samples, while a “separating gel” is the denser bottom layer that actually sorts proteins by size.
People mix them up because both are still called “gel” and sit in the same cassette; the difference is only visible once power is on and bands appear, making the names feel interchangeable.
Key Differences
Stacking gel has low acrylamide and a high pH, so samples pile into a single tight line. Separating gel has higher acrylamide and a lower pH, causing proteins to spread out by molecular weight.
Which One Should You Choose?
You don’t pick one over the other; you cast both. Put stacking gel on top to sharpen the start, then let separating gel do the actual separation. Skipping either ruins the readout.
Examples and Daily Life
Think of stacking gel as the start line of a race that gathers runners, and separating gel as the track where sprinters and joggers naturally spread apart based on speed.
Can I run a gel with only separating gel?
Yes, but bands will smear; the stacking layer keeps them neat.
Do I need different buffers for each?
Usually, the same running buffer covers both layers; no swap is required.
How can I tell them apart after casting?
Stacking gel stays slightly hazy; separating gel looks clearer and sits below.