ANTIBODY-BASED PROTEOME ARRAYS

Gunjan SharmaSaraswathi 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 concentrationRequirements 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 Chemiluminescence

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

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