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Dwuwymiarowa elektroforeza żelowa
Dwuwymiarowa elektroforeza żelowa to metoda separacji białek o wysokiej rozdzielczości, wprowadzona po raz pierwszy przez O'Farrella i Klose w 1975 ro…
Dwuwymiarowa elektroforeza żelowa łączy w sobie dwa wymiary separacji białek: pierwszy oparty na ładunku, a drugi na masie.
Pierwszy wymiar wykorzystuje metodę ogniskowania izoelektrycznego lub IEF. W tym przypadku próbka białka jest ładowana na unieruchomiony gradient pH lub pasek IPG.
Po przyłożeniu prądu elektrycznego białka przemieszczają się w poprzek gradientu pH na pasku, unieruchamiając się w swoim punkcie izoelektrycznym - pH, gdzie białka nie przenoszą ładunku netto.
Następnie pasek IPG jest traktowany SDS i ładowany do żelu poliakrylowego w celu oddzielenia przez drugi wymiar za pomocą SDS-PAGE.
W kierunku prostopadłym do IEF białka rozdzielają się elektroforetycznie w zależności od masy.
Oddzielone białka są następnie wizualizowane po barwieniu.
Dwuwymiarowa elektroforeza żelowa to technika o wysokiej rozdzielczości, która może zidentyfikować podobne białka różniące się nawet jedną naładowaną resztą aminokwasową.
Co więcej, może wykrywać modyfikacje białek wewnątrz komórki lub organelli w różnych warunkach i etapach rozwoju.
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Q1: What are the two dimensions used in two-dimensional gel electrophoresis?
Two-dimensional gel electrophoresis separates proteins by charge in the first dimension and by mass in the second dimension. The first dimension uses isoelectric focusing (IEF) on an immobilized pH gradient strip, where proteins migrate until reaching their isoelectric point—the pH where they carry no net charge. The second dimension uses SDS-PAGE, separating proteins electrophoretically by mass perpendicular to the first dimension.
Q2: How does isoelectric focusing separate proteins on an IPG strip?
Isoelectric focusing uses an immobilized pH gradient (IPG) strip to separate proteins based on their charge. When electric current is applied, amphoteric protein molecules migrate across the pH gradient until each protein reaches its isoelectric point, where it carries no net charge and becomes immobilized. This charge-based separation is the first dimension of two-dimensional gel electrophoresis.
Q3: What is the purpose of treating the IPG strip with SDS before the second dimension?
After isoelectric focusing, the IPG strip is treated with sodium dodecyl sulfate (SDS) buffer to prepare proteins for mass-based separation. SDS coats the uncharged proteins with a negative charge, enabling them to migrate uniformly through the polyacrylamide gel. Glycerol in the buffer reduces electroendosmosis, facilitating protein transfer from the first to the second dimension.
Q4: Why is two-dimensional gel electrophoresis considered a high-resolution technique?
Two-dimensional gel electrophoresis achieves high resolution by separating proteins along two independent parameters: charge and mass. This dual-dimension approach can identify similar proteins differing by even one charged amino acid residue. The technique also detects protein modifications within cells or organelles during different conditions and developmental stages, providing detailed protein analysis.
Q5: What buffer components are used to prepare biological samples for isoelectric focusing?
Biological samples are prepared using a buffer containing urea, dithiothreitol, detergents, and ampholytes. Urea and detergent solubilize and denature proteins, while dithiothreitol, a reducing agent, cleaves disulfide bonds. Ampholytes solubilize proteins and maintain pH, while the buffer rehydrates the IPG strip to help it absorb proteins before charge-based separation.
Q6: What are the limitations of two-dimensional gel electrophoresis for protein analysis?
Two-dimensional gel electrophoresis is unsuitable for distinguishing highly hydrophobic proteins, proteins with high molecular mass, or those present in very low quantities per cell. Additionally, narrower pH gradient IPG strips (such as pH 4-7) detect proteins with higher accuracy than broader-range strips (pH 3-10), requiring careful selection based on sample composition.
Q7: How are separated proteins visualized after two-dimensional gel electrophoresis?
Separated proteins are visualized through multiple methods depending on sample preparation. General staining techniques reveal protein bands on the gel. Radiolabeled samples produce autoradiographic images, while fluorescently labeled samples generate fluorescent images. These visualization approaches enable identification and analysis of the separated protein spots on the gel.