Two-Dimensional Electrophoresis System

What is the Two-Dimensional Electrophoresis System?

A two-dimensional electrophoresis system separates proteins first by isoelectric point in an immobilised pH gradient strip and then by molecular weight in a polyacrylamide gel, resolving thousands of protein spots from complex lysates for comparative proteome analysis.

Overview

The workflow has two stages. In the first dimension, an immobilised pH gradient strip is rehydrated with the sample, and isoelectric focusing is performed under increasing voltage up to several thousand volts until proteins reach the pH at which they carry no net charge. In the second dimension, the strip is equilibrated with DTT and iodoacetamide and placed on top of a polyacrylamide slab gel, where SDS-PAGE separates proteins by molecular weight at right angles to the first dimension. Hundreds to thousands of spots are visualised by Coomassie, silver or fluorescent staining, and images are aligned and compared across samples. Maximum loading is typically 100–1,000 µg of protein per strip for analytical gels, with preparative formats accepting more. Reproducibility depends on consistent sample preparation, focusing conditions and gel casting.

How does it work?

In SDS-PAGE, the sample is denatured with sodium dodecyl sulfate and a reducing agent so that proteins unfold and acquire a uniform negative charge proportional to their length. When an electric field is applied, all proteins migrate toward the anode, and the polyacrylamide mesh sieves them so that migration distance depends mainly on molecular weight. A discontinuous buffer system with stacking and resolving gel layers concentrates proteins into a sharp band before separation. Isoelectric focusing instead uses a pH gradient and separates proteins according to their isoelectric point, where net charge is zero and migration stops. Two-dimensional electrophoresis combines the two, separating by charge first and by size second, which resolves thousands of protein spots. Capillary systems perform SDS or charge separation in a narrow capillary with on-line absorbance or fluorescence detection, replacing staining with quantitative detection.

What are the key features?

• Isoelectric focusing unit for 7 to 24 cm immobilised pH gradient strips
• Gradient or multi-step focusing programs up to 10,000 V
• Second-dimension tank for large format slab gels
• Strip equilibration trays with controlled rocking
• Cooling plate to maintain focusing temperature below 20 °C
• Software support for spot detection, matching and quantification

What are the technical specifications?

A typical two-dimensional electrophoresis system configuration offers strip lengths of 7, 11, 13, 18 or 24 cm, pH gradients of pH 3–10, 3–11 non-linear, 4–7 or 6–11, maximum focusing voltage of up to 10,000 V, focusing current limit of up to 100 µA per strip, sample load per strip of 100–1,000 µg protein (analytical) and second-dimension gel size of approx. 200 x 200 mm. Values are indicative of the product class; the exact figures for each configuration are confirmed on the quotation and the calibration documents supplied with the instrument.

Which applications is it used for?

The Two-Dimensional Electrophoresis System is used in differential proteome expression studies, post-translational modification profiling, biomarker discovery in tissue or plasma and protein complex and isoform analysis. Across the wider Protein Electrophoresis class, laboratories also deploy it for protein purity and molecular weight determination, recombinant protein expression analysis and two-dimensional proteome profiling.

Which standards and certifications apply?

Relevant standards and methods include USP <1057>, Ph. Eur. 2.2.31, Ph. Eur. 2.2.34, IEC 61010-1, ISO 17025 and ISO 15189. Certification candidates for this model are CE, ISO 9001 manufacturing and IEC 61010-1 safety; each is to be confirmed for the specific configuration before shipment, and certificates are supplied only where they have been issued.

Acrylamide is a neurotoxin and suspected carcinogen, so powder handling and gel casting should be performed in a fume hood or with ready-to-use precast gels, and gloves must be worn. Buffer pH and ionic strength affect stacking and resolution, so working buffers should be prepared fresh and not reused excessively. Consistent sample loading, reducing agent freshness and heating time determine how sharp bands appear. Gels should be run within the recommended voltage and current limits, and temperature control matters for high-voltage runs because Joule heating distorts bands. Staining and destaining protocols must be standardised for comparative densitometry, and images used for purity calculations should be captured in the linear range of the camera. Keep records of gel percentage, running conditions and staining for traceability.

How do I select the right Two-Dimensional Electrophoresis System?

Select a two-dimensional system when complex protein mixtures must be resolved into individual species and compared across conditions. Specify strip lengths and pH ranges, maximum voltage and current, second-dimension gel size, cooling capacity and the image analysis software available.

How does it compare with other Protein Electrophoresis variants?

Start with the purpose. For routine purity and molecular weight, a vertical SDS-PAGE system with 1 mm gels and 10–15 wells covers most needs and is inexpensive. Mini systems reduce buffer and sample consumption, run faster and are convenient for immunoblotting, at the cost of lower resolution for closely spaced bands. Multichannel systems process 6 to 12 gels in one tank when screening many samples. Two-dimensional electrophoresis is chosen when complex proteomes must be resolved, and it requires an isoelectric focusing first dimension plus consistent sample preparation. Capillary systems give numerical purity results and are preferred where regulated release testing, low sample volume or full automation is required. Check gel percentage and gradient availability, maximum voltage and current, cooling provisions, glass plate compatibility and whether precast gels are supported.