Finish up array applications Move on to proteomics Protein microarrays

1. Finish up array applications • Move on to proteomics • Protein microarrays

2. Applications of DNA microarrays • Monitor gene expression • Study regulatory networks • Drug discovery - mechanism of action • Diagnostics - tumor diagnosis • etc. • Genomic DNA hybridizations • Explore microbial diversity • Whole genome comparisons - genome evolution • Identify DNA binding sites • Diagnostics - tumor diagnosis • ?

3. Identification of DNA regions bound by a protein. • Compare a wild-type strain to a ∆gene (DNA-binding protein). • Do not need any prior knowledge of the sequence the protein binds. Iyer et al. 2001 Nature, 409:533-538

4. Identifying replication origins in yeast • Only 5% of the genome previously screened for replication origins. • Used known replication initiation factors to perform ChIP/chip analysis • Identified hundreds of additional replication origins in a single experiment.

6. DNA diagnostics • Uses of microarrays is cancer research and diagnosis. • 2733 papers published on microarrays and cancer • 1038 papers published on microarrays, gene expression, cancer diagnosis • 0 since 1997 • Gene expression profiling • Identify genes involved in cancer diagnosis. • Identify gene expression patterns that are associated with disease outcome. • Gene content analysis • Identify genomic regions that are lost or amplified in tumors.

7. Gene expression and cancer • Hierarchical clustering • Method for analyzing microarray data • Gene level analysis • Experiment level analysis

8. Vant Veer et al. 2002 Nature

9. Why study proteins? • They are the machines that make cells function. • RNA levels do not always accurately predict protein levels. • Often processes are regulated at the transcriptional level. • Some processes are controlled post-transcriptionally. • Most often proteins are the targets of drugs.

10. Proteomics -large scale analysis of proteins • Protein levels - Determining the abundance of proteins in a sample. • 2D gel electrophoresis, mass spectrometry, protein microarrays • Interacting proteins - determining which proteins come together to form functional complexes. • Yeast 2-hybrid, affinity purification • Subcellular localization - site of localization can often provide clues to the function of a protein. • GFP tagging, immunofluorescence microscopy. • Protein activity - investigating the biochemical activities of proteins. • Structural genomics - high-throughput analysis of the protein structure

11. From www.probes.com

12. Proteins • Primary structure - sequence • Searching databases • Identifying functional domains • Secondary and tertiary structure - 3D folding of proteins. • Proteins have unique 3D structures • Identify functional domains • VAST - online structural tool from NCBI

13. Western Blot • Determine the presence and level of a protein in a cell lysate. • http://web.mit.edu/esgbio/www/rdna/rdna.html - review of Northern, Western, and Southern blots.

14. Monitoring protein levels - large scale • 2D gel electrophoresis • Old technology - not as useful for lowly expressed proteins. • Mass spectrometry • Many new techniques for protein detection and quantitation being developed. • Protein microarrays • Many developing technologies

15. Protein microarrays • Analysis of thousands of proteins at one time. • Many different types • Antibody arrayed - detect many proteins • Proteins arrayed - detect interacting proteins • Proteins arrayed - detect interacting small molecules • Etc.

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

17. Protein:protein interactions

18. Protein activity arrays

19. Small molecule arrays

20. Why bother with DNA microarrays? • Protein microarrays are not as robust • DNA is DNA - all features will behave similarly under single hybridization conditions. • Proteins are unique - will behave differently. • Protein microarrays are costly • $500-1000 per antibody • $10 per oligo • Used for different purposes