Protein Microarrays

A protein microarray (or protein chip) is a high-throughput method used to track the interactions and activities of proteins, and to determine their function, and determining function on a large scale. Its main advantage lies in the fact that large numbers of proteins can be tracked in parallel.

1. Romil Jayantilal Mistry Roll No. 10 M.Sc. B.T. (8 th Sem ) Protein Microarrays

2. Conceptually, protein microarrays are similar to DNA microarrays. They consist of large numbers of proteins individually immobilized in known positions on the coated surface of glass slide or silicon chip. The proteins arrayed can be antibodies specific for each protein in an organism, purified recombinant proteins, or short synthetic peptides. There are many ways of attaching a protein to a support surface. The major objective of any coupling system is maintenance of protein structure and function. Protein Microarray

3. Some systems bind proteins to a chemical group that coat the surface of the support. With other protocols, recombinant proteins are prepared with a short amino acid sequence (tag) at N or C terminus that bind to a recognition sequence on the support. In this case, all the protein molecules are uniformly oriented. Instead of spotting proteins on a flat surface, some microarrays are engineered with tiny depression ( nanowells ) that keep each protein moist and prevent mixing with adjacent proteins.

4. The purpose of protein microarray analyses is to detect, on a large scale, the molecules that a protein interacts with. These interacting molecules can be other proteins, nucleic acid sequences, or low molecular-weight compounds. Protein populations from different samples can be compared, for example, in control versus treated samples or in normal versus diseases tissues.

5. Direct labeling – to label the test samples directly with a fluorescent dye and then detect the labeled molecules that bind to the proteins of a microarray with a laser scanner. Two-dye strategy (e.g. Cy3 or Cy5) can be used to compare proteins in two different sample on a single array. Sandwich style – the sample molecules are biotinylated , and after the initial incubation, a streptavidin-fluorescent-dye conjugate that binds to biotin to facilitated the detection of sample molecules is applied. Protein Microarray - Visualizing

6. Templin et al. 2002 Trend in Biotch. Vol 20

7. Protein array detection method

8. http://www.nature.com/nature/journal/v422/n6928/images/nature01512-f1.2.jpg

9. Protein microarrays Many different types Antibody arrayed - detect many proteins Proteins arrayed - detect interacting proteins Proteins arrayed - detect interacting small molecules , etc .

10. Protein:Protein Interactions

11. Protein activity arrays

12. Small molecule arrays

13. Cytokine antibody microarrays are used to examine cytokines in both normal and diseased states, and from a variety of sources after various treatments . A sandwich immunoassay is often used to detect cytokines that bind to immobilized antibodies . After the microarray is treated, biotynylated cytokine antibodies are added and bind to the corresponding captured cytokine. For visualization, a streptavidin-fluorescent-dye conjugate attaches to the biotin of the secondary antibody. The signals are detected with a laser scanner. Analytical microarray

14. Cytokine Antibody M icroarray

15. Analytical A ntibody Microarray

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