A brief explanation about antibody based proteome arrays. The mechanism and functional units behind it. The labelling technology used in proteome array experiments. the detection and data analysis of the proteome array is also explained.

1. ANTIBODY-BASED PROTEOME
ARRAYS
Gunjan Sharma
Saraswathi Rajakumar
10th November 2016

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
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