Isoelectric Focusing and IPG Strips Explained: The 2D Gel First Dimension
Isoelectric focusing (IEF) is the first dimension of 2D gel electrophoresis and the one that gives the technique its resolving power. It separates proteins by their isoelectric point, the pH at which a protein carries no net charge, and it can resolve two proteins that differ by a single charge. The method became reproducible enough for proteomics only when the pH gradient was fixed into the gel itself, in the immobilized pH gradient (IPG) strip. This article explains the principle of isoelectric focusing, what an IPG strip is and why it replaced carrier ampholytes, how to choose a strip’s pH range and length, how to rehydrate and focus it, and how to read the common IEF problems from the gel.
What is isoelectric focusing?
Isoelectric focusing is electrophoresis through a pH gradient. A protein placed in the gradient carries a net charge that depends on the local pH: positive where the pH is below its isoelectric point, negative where it is above. Under an electric field, the protein migrates toward the electrode of opposite charge, and as it moves it passes through changing pH. When it reaches the pH equal to its isoelectric point its net charge is zero and it stops. If it diffuses away, it regains charge, in the direction that pulls it back, so proteins are focused into sharp bands rather than spread by diffusion. This self-correcting behavior is what makes IEF the highest-resolution separation of proteins by charge.
In 2D gel electrophoresis, IEF is run in a narrow strip of gel, and the focused strip is then laid across the top of an SDS-PAGE slab so that the proteins are separated by molecular weight at right angles. O’Farrell introduced the combination in 1975, resolving 1,100 components from Escherichia coli, showing that proteins differing in a single charge could be separated, and estimating that up to 5,000 proteins could be resolved on one gel. Our guide to 2D electrophoresis covers the whole workflow; this page is about the first dimension.
The isoelectric point
The isoelectric point (pI) of a protein is set by its ionizable groups: the side chains of aspartate, glutamate, histidine, cysteine, tyrosine, lysine and arginine, and the terminal amine and carboxyl groups. Acidic proteins, rich in aspartate and glutamate, have low pI values; basic proteins, rich in lysine and arginine, have high ones. Most cellular proteins fall between pH 4 and 7, which is why a pH 4 to 7 strip is the most common choice for a first survey, and why the acidic and basic ends of the proteome need dedicated strips.
Post-translational modifications shift the pI. Phosphorylation adds negative charge and moves a protein toward the acidic end by a fraction of a pH unit per phosphate; deamidation, acetylation and glycosylation all change it as well. On a 2D gel these shifts appear as horizontal trains of spots at the same molecular weight, each a differently modified form of one protein. This is the reason 2D gels can distinguish isoforms and modification states that a peptide-based method cannot see directly, and it is the reason a pI shift is an experimental result and not an artifact.
Each protein migrates until the local pH equals its pI, where its net charge is zero. A protein that drifts away regains charge and is pulled back, which is why IEF bands are so sharp.
Carrier ampholytes vs immobilized pH gradients
In O’Farrell’s original method, the pH gradient was generated by carrier ampholytes: a mixture of small, soluble amphoteric molecules with a range of pI values, which arrange themselves under the field into a gradient in a tube gel. The method worked, but the gradient was not stable. The ampholytes themselves migrate, so the gradient drifts toward the cathode over the course of the run (“cathodic drift”), the gradient shape depends on the ampholyte batch, and the loading capacity is limited. Gels from different days, and from different labs, did not match well.
Bjellqvist, Righetti, Görg and colleagues solved this in 1982 by fixing the gradient into the gel. Their Immobiline reagents are acrylamide derivatives carrying buffering groups, which are copolymerized into the polyacrylamide matrix so that the pH at every point is set by the covalently bound buffers and cannot move. The gels are cast like pore-gradient gels, with a light and a heavy solution adjusted to different pH values, and the available Immobiline species can generate any narrow linear gradient between pH 3 and 10. Their 1982 paper lists the advantages: cathodic drift is completely abolished, resolution and loading capacity are higher, conductivity and buffering capacity are uniform, and the ionic strength is known and controlled.
| Property | Carrier ampholyte IEF | Immobilized pH gradient (IPG) |
|---|---|---|
| How the gradient forms | Soluble ampholytes arrange themselves under the field | Buffering groups copolymerized into the gel; gradient is cast in |
| Gradient stability | Drifts toward the cathode during the run | Fixed; no cathodic drift |
| Reproducibility between runs | Depends on ampholyte batch and run time | High; same gradient every time |
| Loading capacity | Limited | Higher; suits micropreparative loads |
| Resolution | Good | Higher; narrow gradients resolve 0.001 pH units |
| pH range | Broad gradients; extremes unstable | Any narrow linear gradient pH 3 to 10 at introduction; now pH 2.5 to 12 |
| Format | Tube gels cast in the lab | Dried strips, commercially manufactured |
| Role today | Largely historical; some specialist uses | Standard first dimension for 2D gels |
What an IPG strip is
An IPG strip is a thin layer of polyacrylamide gel, with the immobilized gradient cast into it, bonded to a plastic backing sheet and supplied dried. The backing makes it possible to handle a gel that would otherwise be a fragile ribbon, and drying makes it possible to manufacture strips in bulk, store them frozen, and rehydrate them with the sample when they are needed. Strips are sold by length (typically 7, 11, 13, 17, 18 and 24 cm) and by pH range (broad, such as 3 to 10; medium, such as 4 to 7; and narrow, one-unit ranges).
Görg and colleagues developed the IPG method into the standard 2D workflow, sometimes called IPG-Dalt, through the late 1980s and 1990s, and their 2000 and 2004 reviews are the reference protocols. By 2004 gradients extended from pH 2.5 to 12, narrow-range strips could resolve proteins whose pI differed by 0.001 pH units, and the combination of wide-range overview gels with narrow-range “zoom” gels had become the standard way to see both the whole proteome and its crowded regions.
Choosing a pH range and strip length
Two decisions set what a 2D gel can show.
pH range. A broad strip (pH 3 to 10) puts the whole proteome on one gel and is the right first survey of an unfamiliar sample. A medium strip (pH 4 to 7, or 5 to 8) spreads the region where most proteins fall over the full width of the gel and resolves proteins that would overlap on a broad strip. Narrow strips (one pH unit, or even 0.5) are zoom gels for a region that is crowded or that contains the proteins you care about. Görg and colleagues describe using wide IPGs up to pH 12 for overview patterns and narrow IPGs as zoom-in gels for optimum resolution and detection of minor components. Basic proteins (pH above 9) need dedicated basic-range strips and modified running conditions.
Strip length. Longer strips spread the same pH range over more distance, which increases resolution and loading capacity at the cost of longer focusing times and a larger second-dimension gel. Short strips (7 cm) suit method development and screening; 17, 18 and 24 cm strips suit analytical and preparative gels where resolution matters. Rehydration volume and volt-hours scale with length, as the tables below show.
| Strip length | Rehydration volume | Maximum focusing voltage | Total volt-hours |
|---|---|---|---|
| 7 cm | 125 µl | 4,000 V (250 V for 20 min, then a 2 h linear ramp to 4,000 V, then hold) | 10,000 Vh |
| 11 cm | 185 µl | 8,000 V | 20,000 Vh |
| 17 cm | 300 µl | 10,000 V | 40,000 Vh |
Sample loading: rehydration vs cup loading
There are two ways to get the sample into the strip, and Görg and colleagues’ 2000 review weighs them against each other.
In-gel rehydration dissolves the sample in the rehydration buffer (urea, thiourea, a non-ionic or zwitterionic detergent, a reducing agent and carrier ampholytes) and lets the dried strip swell in it, so that the protein enters the gel as the strip rehydrates. It is simple, it accommodates large volumes and therefore large protein loads, and it distributes the sample along the whole strip. Passive rehydration takes 11 to 16 hours; some protocols apply a low voltage during rehydration (“active rehydration”) to help large proteins enter the gel.
Cup loading applies the sample in a small cup at one end of the rehydrated strip, usually the anodic end, so that the proteins enter the gel under the field. It is preferred for basic-range strips, where in-gel rehydration performs poorly, and it can give sharper spots for some samples, but the load is limited by the cup volume and the sample enters the gel at a single point.
Görg and colleagues’ guidance is that the choice depends on the pH interval: in-gel rehydration for most acidic and medium ranges, cup loading for basic ranges and for narrow ranges where the sample must be concentrated at one end.
Whichever method is used, the sample must be salt-free or nearly so. Salt carries current, heats the strip and stalls focusing; the classic symptom is a strip that never reaches its target voltage. Protein loads depend on the strip length, the pH range and the stain: silver and fluorescent stains need tens of micrograms on an analytical strip; Coomassie needs several hundred; preparative loads for spot picking run to a milligram on long strips.
The focusing program: voltage and volt-hours
Focusing is run in a dedicated IEF unit that holds the strips under a layer of mineral oil, to stop evaporation and carbon dioxide absorption, and controls temperature, usually at 20 °C. The program steps the voltage up in stages.
The first stage is low voltage (a few hundred volts) with a current limit, while the sample is still conductive with residual salt and the ampholytes are entering the gel. The second stage ramps the voltage to the maximum for the strip length. The final stage holds at maximum voltage until a set number of volt-hours has accumulated. Volt-hours (voltage multiplied by time) is the measure of focusing, because a protein’s progress to its pI depends on the field it has experienced and for how long; a strip that ran at lower voltage needs more time to reach the same volt-hours.
Under-focusing leaves proteins short of their pI and gives horizontal streaks or smeared spots; over-focusing, especially of basic ranges, can cause proteins to drift or precipitate and gives loss of spots and a weak basic end. The Bio-Rad figures in the table above (10,000 Vh for a 7 cm strip, 20,000 for 11 cm, 40,000 for 17 cm) are a starting point; the right value for a given sample is found by running a series and choosing the one with the sharpest spots. Focused strips can go straight to equilibration or be stored frozen at −80 °C.
After focusing: equilibration and the second dimension
Before the strip can run in the second dimension, the focused proteins must be converted from the charged, folded state in which they focused to the uniformly negative, unfolded state that SDS-PAGE needs. The strip is equilibrated in a buffer containing SDS, urea and glycerol, first with a reducing agent (typically DTT) to break disulfide bonds and then with iodoacetamide to alkylate the freed thiols so they cannot re-form. Each step takes 10 to 15 minutes. The strip is then laid along the top of the SDS-PAGE slab, sealed in place with agarose, and run. The guide to 2D electrophoresis covers the second dimension and the steps that follow.
Troubleshooting IEF problems
Most first-dimension problems can be read from the 2D gel, because IEF faults produce distortions along the horizontal (pI) axis while second-dimension faults produce them along the vertical axis. Görg and colleagues include a troubleshooting guide in their 2000 review; the table below summarizes the common cases.
| Symptom on the 2D gel | Likely IEF cause | Fix |
|---|---|---|
| Horizontal streaking across the gel | Under-focusing; salt or other ionic contaminants; insufficient solubilization | Increase volt-hours; desalt or precipitate the sample; add thiourea and check detergent |
| Horizontal streaking of a few abundant spots | Overloading; protein precipitating at its pI | Reduce load; use a longer strip or a narrower range |
| Weak or empty basic end | Basic proteins lost by drift or reduction problems | Use cup loading at the anode for basic strips; add excess DTT or use a different reductant; shorten focusing |
| Weak or empty acidic end | Sample applied at the wrong end; acidic proteins lost in rehydration | Check loading position; try in-gel rehydration so the sample is distributed along the strip |
| Spots missing at high molecular weight | Large proteins failed to enter the strip | Active rehydration at low voltage; cup loading |
| Strip never reaches target voltage | Excess salt or ionic detergent in the sample | Desalt; keep SDS in the sample below the level tolerated; replace electrode wicks |
| Burnt or brown strip ends | Current too high; salt; dry electrode contact | Limit current per strip; desalt; wet the wicks |
| Vertical streaks | Second-dimension problem (poor equilibration, nucleic acids, dust), not IEF | Extend equilibration; nuclease treatment; filter samples |
| Whole pattern shifted left or right compared with other gels | Different strip lot, focusing time or temperature | Standardize; image alignment will correct the residual shift |
The last row is the bridge to analysis: even with IPG strips and a standard program, no two gels focus identically, and the residual horizontal shift is one of the things image alignment removes.
What IEF means for image analysis
Every choice made in the first dimension shows up in the image. Strip length sets the width of the gel and therefore the pixel size a scanner needs; the pH range sets whether the pattern is a broad overview or a crowded zoom in which spots overlap; focusing quality sets whether spots are sharp enough to be detected and quantified cleanly; and run-to-run drift is what alignment has to correct before gels can be compared.
SameSpots handles the analysis side of this. It aligns every image to a reference at the pixel level, correcting the horizontal shifts that remain after focusing as well as the vertical ones from the second dimension, then detects one spot pattern across the whole experiment so that every spot is measured on every gel. Because pI and molecular weight markers can be defined within the gel images, SameSpots calibrates the pI axis so that each spot’s position is reported in pH units, which turns a horizontal train of spots into a set of measured pI shifts. Our articles on 2D gel image alignment and on spot detection and quantification cover the next steps.
Frequently asked questions
Q: What is isoelectric focusing?
A: Electrophoresis through a pH gradient in which each protein migrates until it reaches the pH equal to its isoelectric point, where it has no net charge and stops. Proteins that diffuse away regain charge and are pulled back, so they focus into sharp bands.
Q: What is the isoelectric point of a protein?
A: The pH at which a protein carries no net charge. It is set by the protein’s ionizable amino acid side chains and termini and is shifted by modifications such as phosphorylation.
Q: What is an IPG strip?
A: A dried strip of polyacrylamide gel on a plastic backing with an immobilized pH gradient cast into it. The gradient is formed by buffering groups covalently bound to the gel, so it cannot drift. Strips are rehydrated with sample and focused to give the first dimension of a 2D gel.
Q: What is the difference between carrier ampholytes and IPG strips?
A: Carrier ampholytes are soluble molecules that form a gradient under the field and drift during the run. In an IPG the gradient is fixed into the gel. IPGs abolish cathodic drift, give higher resolution and loading capacity, and are reproducible from run to run.
Q: Which pH range IPG strip should I use?
A: A broad strip (pH 3 to 10) for a first survey; a medium strip (pH 4 to 7) for most cellular proteomes; narrow one-unit strips to zoom into a crowded region; dedicated basic-range strips for proteins above pH 9.
Q: How long should an IPG strip be rehydrated?
A: Passive rehydration takes 11 to 16 hours, typically overnight, in volumes of 125 µl for a 7 cm strip, 185 µl for 11 cm and 300 µl for 17 cm. Active rehydration at low voltage helps large proteins enter the gel.
Q: What are volt-hours in isoelectric focusing?
A: Voltage multiplied by time, the measure of how much focusing a strip has received. Typical totals are 10,000 Vh for a 7 cm strip, 20,000 Vh for 11 cm and 40,000 Vh for 17 cm; the best value for a sample is found by trial.
Q: What causes horizontal streaking on a 2D gel?
A: Usually a first-dimension problem: under-focusing, salt or ionic contaminants in the sample, incomplete solubilization, or overloading. Desalting, more volt-hours and a lower load are the usual fixes.
Q: Why is my strip not reaching its target voltage?
A: Too much salt or ionic detergent in the sample is carrying the current. Desalt or precipitate the sample and check the electrode wicks.
Q: How is isoelectric focusing used in 2D gel electrophoresis?
A: It is the first dimension. Proteins are focused by pI on an IPG strip, the strip is equilibrated in SDS buffer, and it is laid on an SDS-PAGE gel so that the proteins are separated by molecular weight at right angles to the first separation.
References
1. Bjellqvist B, Ek K, Righetti PG, Gianazza E, Görg A, Westermeier R, Postel W. Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications. J Biochem Biophys Methods. 1982;6(4):317-39. https://doi.org/10.1016/0165-022X(82)90013-6
(Source for: buffering groups covalently linked to the matrix; Immobiline monomers; casting like pore-gradient gels with light and heavy solutions; any narrow linear gradient between pH 3 and 10; cathodic drift abolished; higher resolution and loading capacity; uniform conductivity and buffering capacity; controlled ionic strength.)
2. O’Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975;250(10):4007-21. https://doi.org/10.1016/S0021-9258(19)41496-8
(Source for: IEF by pI in the first dimension and SDS by molecular weight in the second; 1,100 components resolved from E. coli; up to 5,000 proteins; proteins differing in a single charge resolved.)
3. Görg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, Weiss W. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis. 2000;21(6):1037-53. https://doi.org/10.1002/(SICI)1522-2683(20000401)21:6<1037::AID-ELPS1037>3.0.CO;2-V
(Source for: cup loading versus in-gel rehydration and their dependence on pH interval; wide IPGs up to pH 12 for overview patterns and narrow IPGs for zoom-in gels; the IPG-Dalt protocol; the troubleshooting guide.)
4. Bio-Rad Laboratories. ReadyPrep 2-D Starter Kit Instruction Manual, Bulletin 4110009A. https://www.bio-rad.com/webroot/web/pdf/lsr/literature/4110009A.pdf
(Source for: rehydration volumes by strip length; passive rehydration 11 to 16 hours; focusing voltage programs and volt-hour totals; equilibration buffers and times.)
5. Görg A, Weiss W, Dunn MJ. Current two-dimensional electrophoresis technology for proteomics. Proteomics. 2004;4(12):3665-85. https://doi.org/10.1002/pmic.200401031
(Source for: IPG range pH 2.5 to 12; narrow IPG resolution of 0.001 pH units; strip formats; loading guidance by range and stain; basic-range handling.)
6. TotalLab. SameSpots 2D gel analysis software. https://totallab.com/software/2d-gel-analysis-software/
(Source for: pixel-level alignment; one spot pattern across the experiment; pI and MW calibration from markers defined in the images.)
Statements about pI chemistry (which side chains ionize, the
direction of the phosphorylation shift) are textbook biochemistry and carry no numerical claims. The troubleshooting table combines reference 3 with practitioner guidance.
From focused strip to finished statistics
Once the gels are run, SameSpots aligns them, calibrates the pI axis from your markers, detects one spot pattern across the whole experiment and runs the statistics, with a typical gel taking under five minutes. Request a trial and analyze your own gels.