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Selecting The Optimal Gel Percentage in Electrophoresis

Low visibility, fuzzy, and poorly separated bands, as well as inconsistent migration are common gel electrophoresis problems that can reduce yield and generate unreliable quantitative data, wasting valuable time and resources. Choosing the optimal percentage gel is critical in avoiding these problems and streamlining your workflow.

Acrylamide Gels: Protein Separation

Polyacrylamide gels are characterized by two parameters: total acrylamide percentage (%T, in g/100 ml) and weight percentage of cross-linker (%C). Varying the ratio of these parameters changes the pore size to optimize separation and resolution of target proteins. The smaller the size of the protein of interest, the higher the percentage of acrylamide/bis (%T).

To enhance resolution and form tight bands, a discontinuous buffer system is used for protein separation.This is accomplished by using a gel separated into two sections: a large-pore stacking gel on top of a small-pore resolving gel.

  • Stacking gels are 4%T and are made with a buffer of pH 6.8.
  • Resolving gels can be made as a single percentage (typically ranging from 7.5% - 20%) or as a gradient (typically 4-15% and 10-20%) and are made with a buffer of pH 8.8.

Below is a general guide for choosing an appropriate gel percentage based on protein size.

Protein size Gel acrylamide percentage
4–40 kDa Up to 20%
12–45 kDa 15%
10–70 kDa 12.5%
15–100 kDa 10%
25–200 kDa 8%
>200 4-6%
Unknown Sample 4-20% gradient or 8-16% gradient

Pro Tips:

  • Use single-percentage gels to separate proteins with similar molecular weights, with your target protein migrating to the lower half of the gel -- where optimal separation occurs.
  • For samples with a broad range of molecular weights, use gradient gels. Higher molecular weight proteins will be resolved in the upper portion of the gel and lower molecular weight proteins will be separated in the lower portion.
  • For unknown samples use a broad gradient. Once the target protein size range is identified, use a single-percentage gradient for better resolution.
  • Remember, acrylamide is a potent cumulative neurotoxin: wear gloves at all times.

Agarose: Nucleic Acid Separation

Typically a 0.7% to 2% agarose gels are used, but the ideal percentage depends on the fragment size of interest, how good separation needs to be, and if the gel slices will be further processed/cleaned. A higher agarose percentage enhances resolution of smaller bands and a lower agarose percentage gives better resolution and separation of higher molecular-weight bands. For a sample with a wide range of sizes to be separated, start with a 1.20% agarose gel concentration.

Resolution Agarose % (w / v)
1,000 – 30,000 bp 0.50%
800 – 12,000 bp 0.70%
500 – 10,000 bp 1.00%
400 – 7,000 bp 1.20%
200 – 3,000 bp 1.50%
50 – 2,000 bp 2.00%

Pro Tips for resolving fragments of similar size:

  • Use a high percentage gel to (2.00% - 3.00%)
  • Use a larger agarose gel to allow for a longer run time
  • Use a wider/thinner gel comb to enhance the crispness of the bands
  • Avoid high voltage and run electrophoresis slowly for longer
  • Use continuous polyacrylamide gels (no stacking gel) to separate 5 - 500 bp fragments

Get everything you need for electrophoresis with MP Bio’s comprehensive selection of materials to separate DNA, RNA, and proteins.