The first dimension of 2D-PAGE separates proteins by isoelectric point on an immobilised pH gradient strip, and choosing the strip range is the single biggest lever on resolution. This selector matches your protein or proteome pI range to the appropriate broad, medium, or narrow IPG strip.
How it works
A protein focuses at the position in the gradient where the local pH equals its pI, because that is where its net charge is zero and it stops migrating. The rule the selector applies is simple: the strip range must bracket every pI you care about, and the narrower the bracket, the higher the resolution per pH unit:
resolution ∝ strip length / (pH_high − pH_low)For a survey it recommends a broad strip (pH 3–10) that catches most proteins in one run. For a zoom it recommends the narrowest standard strip that still spans your entered pI range, so closely spaced spots separate as far as possible.
Standard IPG strip ranges and their uses
| Strip pH range | Type | Best used for |
|---|---|---|
| pH 3–10 (linear) | Broad | Whole-proteome survey, first experiment on a new sample |
| pH 3–10 (non-linear) | Broad | Better resolution at the extremes (very acidic and very basic) |
| pH 3–11 | Broad | Capturing highly basic proteins missed by 3–10 linear |
| pH 4–7 | Medium | Most cytosolic proteins; high resolution in this common window |
| pH 6–11 | Medium | Basic and very basic proteins |
| pH 4–5, pH 5–6, pH 6–7 | Narrow zoom | High resolution within a 1-pH-unit window |
The choice of linear versus non-linear within the broad range matters: a non-linear gradient compresses the over-represented acidic range (pH 4–6 contains the majority of mammalian cytosolic proteins) and expands the basic end, giving more even spot distribution across the strip.
The pI distribution of common proteomes
The majority of proteins in a typical mammalian cell lysate have pI values between 4 and 7. A second, smaller cluster of more basic proteins (ribosomal proteins, histones, and many nuclear proteins) appears in the pH 8–11 range. Very few proteins have pI at the extremes (below 3.5 or above 11). This bimodal distribution explains why pH 4–7 and pH 6–11 strips are so commonly used together to provide near-complete cytosolic coverage at good resolution.
Effect of post-translational modifications on pI
IEF is run under denaturing conditions (urea, reducing agent), so it resolves the intrinsic pI of the unfolded polypeptide plus its covalent modifications. Key effects:
- Phosphorylation adds negative charge and shifts pI toward acidic, typically by 0.5–2 pH units per phosphate group. A phosphorylated protein appears as a horizontal spot train to the left of its unmodified form.
- Deamidation of Asn/Gln adds negative charge and is a common age-related modification; the modified spot appears more acidic.
- Glycosylation affects mass more than charge in most cases, shifting the spot vertically (second dimension) rather than horizontally.
When your protein of interest is known to be modified, plan for the pI of each modification state to fall within the strip window.
Notes and tips
Standard commercial ranges include broad pH 3–10 and 3–11, medium pH 4–7 and 6–11, and narrow zoom strips such as 4–5, 5–6, and 6–7. Running several overlapping narrow strips is how proteomics labs achieve whole-proteome coverage at high resolution. Remember IEF runs denatured, so use denatured pI values, and expect modified proteins (such as phosphorylated forms) to appear as horizontal spot trains shifted toward lower pI.