Protein Microarrays
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Visit Molecular Station for a great article on Antibody and Protein Microarrays
Protein Chips:
Protein Microarrays can be based on any ligand-binding assay that relies on the formation of product with an immobilized capture molecule and a target molecule present in the solution. These assays can be miniturized and placed on a protein microarray chip.
Protein microarrays are now becoming very popular due to their possible future applications in the study of nucleic acid-protein, protein-protein, ligand-receptor, drug-protein target, and enzyme-substrate interactions.
Types of Capture Molecules for Protein Microarray Chips. To analyze protein interactions with other molecules, protein microarrays can have various types of molecules immobilized on the slide surface to act as capture molecules in the protein microarray assay.
Figure 1 Illustrates the most common protein array, the antibody microarray. Antibodies are spotted onto a glass slide. Two cell lysates with proteins labeled with Cy3 and Cy5 (for example, cancer vs normal) are added as a solution onto the protein array. Signals are detected from the two different signals. Data is computed allowing an analysis of how much dye-labeled protein the antibodies on each spot are binding. This information allows one to determine whether the protein expression is up-regulated in cancer, down-regulated or unchanged.
Figure 2. a) Demonstrates protein arrays which are based on microarray analysis of antigen-antibody interactions. Antigens are spotted onto glass slides. Antibodies which are tagged bind to antigens and emit fluorescent signal (shown as the yellow star) which can then be detected from the spot on the array.
b) Shows a protein microarray composed of spots which act as sandwich immunoassays. Antibodies are spotted onto the chips and cell lysate or a solution is applied to the slide. Antibodies are incubated with the solution, with later washing to remove non-specific binding. The sandwich complexes formed emit a detectable signal from the spot.
c) A protein-protein interaction protein microarray where proteins are spotted onto a protein chip, and labeled proteins are added to the chip and incubated to determine where they may bind and interact. A detectable signal is emitted from binding spots.
d) Nucleic Acid-protein interaction array, (here a DNA-protein interaction chip). Nucleic acids such as DNA are spotted onto a glass slide. This could be transcriptional binding sites for example. Proteins such as fluorescently labeled DNA-binding transcription factors in solution are added to the chip and incubated on the array. A detectable signal is emitted from the DNA spots where the proteins bind.
e) If you have a set of ligands and you want to find the receptors the bind to, you can use a receptor-ligand protein chip as displayed. Receptor proteins are spotted on glass slides. Fluorescently labeled ligands are added to the chip. Binding of ligand to receptors causes the emission of a detectable signal which pinpoints the interacting spots (i.e. which protein did the ligand bind to).
f) To test for enzyme-subrates specificity one can employ an enzyme-substrate protein microarray. This chip is composed of descrete nano-wells in which enzymes are bound. Substrate is added into the nano-wells and signal emission is detected from the nano-wells in order to assay for enzyme function.
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