Name: protein separation by charge and mass
Uploaded: 2023-04-30T11:58:38.000Z
Duration: 1 min 22 s
Channel: JoVE

Two-dimensional gel electrophoresis combines two dimensions for protein separation: first based on charge, and second by mass.
The first dimension uses the method of isoelectric focusing or IEF. Here, the protein sample is loaded on an immobilized pH gradient or IPG strip.
On applying electric current, the proteins move across the pH gradient on the strip, immobilizing at their isoelectric point - the pH, where the proteins carry no net charge.
Next, the IPG strip is treated with SDS and loaded onto a polyacrylamide gel for separation by the second dimension using SDS-PAGE.
In a direction perpendicular to IEF, the proteins separate electrophoretically based on mass.
The separated proteins are then visualized post staining.
Two-dimensional gel electrophoresis is a high-resolution technique that can identify similar proteins differing by even one charged amino acid residue.
Further, it can detect protein modifications inside a cell or organelle during different conditions and developmental stages.

two-dimensional gel electrophoresis

Two-dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis is a high-resolution protein separation method first introduced by O&#39;&#160;Farrell and Klose in 1975. This method involves protein separation by two dimensions, mass and charge, making it more accurate than one-dimensional gel electrophoresis.
The first dimension separation uses the isoelectric focusing or IEF technique performed on immobilized pH gradient (IPG) strips that separate proteins according to their isoelectric points.
Biological samples, such as&#160; cells and tissues, are first prepared for IEF by treatment with a buffer containing urea, dithiothreitol, detergents, and ampholytes. Here, urea and detergent solubilize and denature the proteins. Dithiothreitol is a reducing agent that cleaves disulfide bonds. Ampholytes used in a suitable concentration solubilize the protein and maintain pH. The buffer also rehydrates the IPG strip that helps it absorb the proteins before their charge-based separation.
Proteins are amphoteric molecules with positive and negative charges. The amphoteric protein molecules migrate on the pH gradient of the IPG strip under the electric current. Once the proteins reach their isoelectric point, they immobilize because, at this point, they have no net charge. IPG strips have a fixed pH gradient; for instance, strips with a smaller pH range (for example, pH 4-7) can detect proteins with higher accuracy than those with a broader range (pH 3-10). Once the proteins separate, the IPG strips are treated with sodium dodecyl sulfate (SDS) buffer to proceed to the next phase, i.e., mass-based separation using SDS-PAGE (polyacrylamide gel electrophoresis). SDS covers the uncharged protein on the IPG strips with a negative charge, and glycerol reduces electroendosmosis which helps the transfer of proteins from the first to the second dimension.
Generally, bands on the gel can be visualized upon staining. Sometimes radiolabelled samples are used to obtain an autoradiographic image. Samples capable of producing fluorescent images can also be used.
Two-dimensional gel electrophoresis has gained importance owing to the precision of the outcome results. However, the method is unsuitable for distinguishing highly hydrophobic proteins,&#160; proteins with high molecular mass, or those present in very low quantities per cell.

Two-dimensional gel electrophoresis is a high-resolution protein separation method first introduced by O&#39;&#160;Farrell and Klose in 1975. This method ...

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Cell Biology

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Analyzing Cells and Proteins

KEY TERMS AND CONCEPTS

protein separation by charge and mass

Two-dimensional gel electrophoresis separates proteins by charge first and then by mass. It was first introduced by O’Farrell and Klose in 1975. Because it uses two separating steps, it gives a higher-resolution result than one-dimensional gel electrophoresis.

The first dimension uses isoelectric focusing, or IEF, on immobilized pH gradient, or IPG, strips. Before this step, biological samples such as cells and tissues are treated with a buffer that contains urea, dithiothreitol, detergents, and ampholytes. Urea and detergent help solubilize and denature the proteins, dithiothreitol breaks disulfide bonds, and ampholytes help keep the pH stable.

The buffer also rehydrates the IPG strip so it can absorb the proteins. Proteins are amphoteric molecules, which means they have both positive and negative charges. Under an electric current, they move along the pH gradient until they reach their isoelectric point, where they have no net charge and stop moving. IPG strips with a narrower pH range, such as pH 4-7, can detect proteins more accurately than broader strips like pH 3-10.

After IEF, the IPG strip is treated with an SDS buffer to prepare for the second dimension. SDS, or sodium dodecyl sulfate, gives the proteins a negative charge so they can separate by size in SDS-PAGE, which stands for polyacrylamide gel electrophoresis. Glycerol helps reduce electroendosmosis and supports the transfer from the first dimension to the second.

The protein bands are usually seen after staining. In some cases, radiolabelled samples are used to create an autoradiographic image, or samples that produce fluorescent signals are used. Two-dimensional gel electrophoresis is valued for its precision, but it is not well suited for highly hydrophobic proteins, proteins with very high molecular mass, or proteins present in very low amounts per cell.

Protein Separation by Charge and Mass

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Two-dimensional gel electrophoresis separates proteins by charge first and then by mass. It was first introduced by O’Farrell and Klose in 1975. Because it uses two separating steps, it gives a higher-resolution result than one-dimensional gel electrophoresis. The first dimension uses isoelectric focusing, or IEF, on immobilized pH gradient, or IPG, strips. Before this step, biological samples such as cells and tissues are treated with a buffer that contains urea, dithiothreitol, detergents,...

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key terms and concepts

Learn how two-dimensional gel electrophoresis separates proteins by charge and mass using IEF, IPG strips, and SDS-PAGE for high-resolution results.

Protein Separation by Charge and Mass | JoVE K12

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Protein Separation by Charge and Mass

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