1 / 18

Protein Arrays

Protein Arrays. By: Nicole Therrien. Overview. What Are Protein Arrays? General Scheme Types of Arrays Analytical In-depth Example Functional Overview Example Reverse Phase Overview Example. What Are Protein Arrays?. Similar to DNA microarrays Plate, Probe, Attachment Advantage

badu
Download Presentation

Protein Arrays

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Protein Arrays By: Nicole Therrien

  2. Overview • What Are Protein Arrays? • General Scheme • Types of Arrays • Analytical • In-depth • Example • Functional • Overview • Example • Reverse Phase • Overview • Example

  3. What Are Protein Arrays? • Similar to DNA microarrays • Plate, Probe, Attachment • Advantage • Poor correlation between mRNA and protein expression • Study protein interactions • Protein-Protein • Protein-Ligand • Protein-DNA • Monitor Disease States • Clinical Diagnostics

  4. General Scheme

  5. Types of Arrays • Analytical Microarrays • Functional Microarrays • Reverse Phase Microarrays

  6. Analytical Microarrays • Profiles Mixture of Proteins • Measure Binding Affinity • Specificity • Protein Expression Levels • Most Common • 3 main probe types • Antibodies • Affibodies • Aptamers

  7. Plate Set Up • Choose plate surface • Glass, Silicon • Attachment Method • Random Attachment • Covalent attachment by amines • Aldehyde • Epoxy • Adsorption • Nitrocellulose • Poly-L-Lysine • Acrylamide Gel Pads • Uniform Attachment • Affinity Tag • Nickel Coat & His tag • Streptavidin& Biotin • Spots vs. Wells • Sample incubated on plate with probes

  8. Analytical Microarray Plates • Antibodies • 150 kDa • Standard • Affibodies • non-immunoglobulin-based affinity reagents • Based on Staphylococcus aureus protein A • Alpha-helices • No Disulfide • 6 kDa • Randomization of 13 AA in binding domain

  9. Plates continued • Aptamers • Nucleic Acids • DNA, RNA, etc. • Peptides • Variable loop (10-20 AA) • Protein Scaffold • Bind Protein • Van der Waals Forces, H bonding, Electrostatic Interaction • Highly Specific • Engineered completely in test tube • In vitro selection

  10. Sample Preparation • Sample extracted from cells or tissues • Bio-Rad assay • Labeled • Fluorescent Dye • Cy3/Cy5 via Lysines • Photochemical • Radioisotope • May interfere

  11. Unlabeled • Antibody Sandwich • 2nd antibody with label incubated on top of sample • Surface Plasmon resonance • Measure electromagnetic waves • Angle changes in the order of 0.1° with 1 nm film adsorption • Needs special equipment • Don’t affect protein structure

  12. Detection & Quantification • Scanner • Detects dye • Adjusts for background • Reference spots • Labeled known concentrations • Computational Analysis

  13. Analytical Microarray Example • Individualized Medicine • Aptamers that recognize drug-resistant HIV-1 reverse transcriptase • In vitro selection • Attached with neutravidin & biotin • Incubated w/ M3 or WT HIV-1 RT • 1° Antibody • Rabbit Anti-HIV-1 • 2°Antibody • Goat Anti-rabbit Cy-3 label • Read by microarray scanner

  14. Functional Microarrays • Plates • Full length proteins & protein domains • Functional • Samples • Purified & Labeled • Nucleic Acids • Proteins • Lipids • Small Molecules

  15. Functional Array Example • Protein-Small Molecule Interaction • Plate has whole proteome • Monitor Specificity • Off-target effects

  16. Reverse Phase Microarrays • Plates • Cell Lysate • Sample • Antibodies of interest • Primary • Attach to spots • Secondary • Attach to primary • Labeled • Detect Altered Proteins • Post-translation modification problems • Disease

  17. Reverse Phase Example • Quantitative cell signaling analysis reveals down-regulation of MAPK pathway activation in colorectal cancer • Mitogen-activated protein kinases • Role in colonic cancer • Lysates of WT and Cancerous cells attached via nitrocellulose • Phospho-specific Rabbit antibodies • Staining • Ras mutations in colorectal cancer • Thought to increase MAPK pathway • Cautioned against kinase inhibition therapy

  18. References • Amaratunga, Dhammika, and Javier Cabrera. Exploration and Analysis of DNA Microarray and Protein Array Data. Hoboken, NJ: John Wiley, 2004. • Gullman, Christian, et al. "Quantitative Cell Signalling Analysis Reveals Down-Regulation of MAPK Pathway Activation in Colorectal Cancer." Journal of Pathology 218 (2009): 514-19 • Hall, David et al.. "Protein Microarray Technology." Mechanisms of Ageing and Development 128.1 (2007): 161-67. \ • Li, Na et al. "Aptamers That Recognize Drug-Resistant HIV-1 Reverse Transcriptase." Nucleic Acids Research 36.21 (2008): 6739-751. • "Panorama Antibody Array Frequently Asked Questions." Sigma Aldrich Home. Web. 10 Apr. 2010. <http://www.sigmaaldrich.com/life-science/cell-biology/protein-arrays/protein-arrays-applications/faq.html>. • "Surface Plasmon Resonance - Wikipedia, the Free Encyclopedia." Main Page - Wikipedia, the Free Encyclopedia. Web. 10 Apr. 2010. <http://en.wikipedia.org/wiki/Surface_plasmon_resonance>. • Tao, Sheng-Ce, et al. "Applications of Protein Microarray Technology." Combinatorial Chemistry & High Throughput Screening 10 (2007): 706-18.

More Related