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ANTIBODY-BASED PROTEOME
ARRAYS
Gunjan Sharma
Saraswathi Rajakumar
10th November 2016
Overview
 Why antibody based proteome array?
 Technical Factors and mechanism
 Antibody and Protein array formats
 Labeling-hybridization methods
 Detection and data analysis
 Current technology
 Conclusion
 Reference
Why we need antibody-based
proteome array?
 Gene expression = cancer biology, early detection, monitoring disease
 Gene Expression determined by
DNA array
 High mRNA don’t mean high
Proteome
 Most cellular process carried out
by protein
 Lower sample volumes, antibody concentration
Requirements and eliminates multiple western blotting
Image source: Slideshare
Image source: Alhamdani et al., 2009
Technical Factors
 Array surface – glass coated
with substrate
 antibodies – Mono specific
polyclonal antibody
 sample processing- low
molecular weight serum
proteome, Fractionate
proteins
 signal generation –
Fluorescent dyes or biotin
derivatives
 data analysis – Spot image
analysis tool, pixel density
Image source: Sanchez-Carbayo., 2006
Antibody and Protein
array formats
 Direct = Protein + Fluorophore
 Indirect = Protein+Tag+
Fluorophore
 Sandwich – 2 antibodies =
increased detection
 Antibody coated beads +
sample (protein binding) in
flow cytometer system
 Antibodies in cancer sera
reacts to Tumor associated
antigens
 Reverse Phase – targeting
specific protein
Image source: Sanchez-Carbayo., 2006
 SAPE – Streptavidin-
R-phycoerythrin
 Tyramide amplification
= increase in biotin
 Green orb – species
specific antibody
Pink orb –horse radish
peroxidase
 Oligonucleotide primer
attached with antibiotin
ab
Antibiotin antibody +
biotinylated ab = oligos
extended with circular
DNA
 Gold particle –
resonance light
scattering
Labeling-hybridization methods
Detection and data analysis
Image source – Wikipedia
 Main
Detection
methods-
radioactivity,
fluorescence
and
Chemilumines
cence
Image source –
R&D systems
Current Research
Conclusion
 Proteome array helps targeting the effector molecule of most biochemical
process ( protein)
 Antibody based has features such as lower sample volume and sensitivity.
 Detects multiple proteins simultaneously.
References
Sanchez-Carbayo, M., 2006. Antibody arrays: technical considerations and clinical
applications in cancer. Clinical chemistry, 52(9), pp.1651-1659.
Alhamdani, M.S., Schröder, C. and Hoheisel, J.D., 2009. Oncoproteomic profiling
with antibody microarrays. Genome medicine, 1(7), p.1.
RECALDE, H., 2011. Slideshare. Metodología de la Investigación http://www.
slideshare. net/hector_recalde/mtodologa-de-la-investigac.
https://www.rndsystems.com/
Lv, L.L. and Liu, B.C., 2014. High-throughput antibody microarrays for
quantitative proteomic analysis. Expert review of proteomics.
Questions and Discussion
Thank you for listening:

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Proteome array - antibody based proteome arrays

  • 2. Overview  Why antibody based proteome array?  Technical Factors and mechanism  Antibody and Protein array formats  Labeling-hybridization methods  Detection and data analysis  Current technology  Conclusion  Reference
  • 3. Why we need antibody-based proteome array?  Gene expression = cancer biology, early detection, monitoring disease  Gene Expression determined by DNA array  High mRNA don’t mean high Proteome  Most cellular process carried out by protein  Lower sample volumes, antibody concentration Requirements and eliminates multiple western blotting Image source: Slideshare
  • 4. Image source: Alhamdani et al., 2009 Technical Factors  Array surface – glass coated with substrate  antibodies – Mono specific polyclonal antibody  sample processing- low molecular weight serum proteome, Fractionate proteins  signal generation – Fluorescent dyes or biotin derivatives  data analysis – Spot image analysis tool, pixel density
  • 5. Image source: Sanchez-Carbayo., 2006 Antibody and Protein array formats  Direct = Protein + Fluorophore  Indirect = Protein+Tag+ Fluorophore  Sandwich – 2 antibodies = increased detection  Antibody coated beads + sample (protein binding) in flow cytometer system  Antibodies in cancer sera reacts to Tumor associated antigens  Reverse Phase – targeting specific protein
  • 6. Image source: Sanchez-Carbayo., 2006  SAPE – Streptavidin- R-phycoerythrin  Tyramide amplification = increase in biotin  Green orb – species specific antibody Pink orb –horse radish peroxidase  Oligonucleotide primer attached with antibiotin ab Antibiotin antibody + biotinylated ab = oligos extended with circular DNA  Gold particle – resonance light scattering Labeling-hybridization methods
  • 7. Detection and data analysis Image source – Wikipedia  Main Detection methods- radioactivity, fluorescence and Chemilumines cence
  • 8. Image source – R&D systems Current Research
  • 9. Conclusion  Proteome array helps targeting the effector molecule of most biochemical process ( protein)  Antibody based has features such as lower sample volume and sensitivity.  Detects multiple proteins simultaneously.
  • 10. References Sanchez-Carbayo, M., 2006. Antibody arrays: technical considerations and clinical applications in cancer. Clinical chemistry, 52(9), pp.1651-1659. Alhamdani, M.S., Schröder, C. and Hoheisel, J.D., 2009. Oncoproteomic profiling with antibody microarrays. Genome medicine, 1(7), p.1. RECALDE, H., 2011. Slideshare. Metodología de la Investigación http://www. slideshare. net/hector_recalde/mtodologa-de-la-investigac. https://www.rndsystems.com/ Lv, L.L. and Liu, B.C., 2014. High-throughput antibody microarrays for quantitative proteomic analysis. Expert review of proteomics.
  • 11. Questions and Discussion Thank you for listening: