Protein array

1. PROTEIN MICROARRAY
DR.MUMTAZ ALI NAREJO
PhD SCHOLAR , SALU KHAIRPUR

2. OUTLINES
• Introduction
• History
• Preparation
• Methods
• Types
• Detection
• Applications
• Challanges

3. INTRODUCTION
• a high-throughput method used to track the
interactions and activities of proteins, and to
determine their function, and determining
function on a large scale
• large numbers of proteins can be tracked in
parallel
• The chip consists of a support surface such as a
glass slide, nitrocellulose membrane, bead,
or microtitre plate, to which an array of capture
proteins is bound

4. • Probe molecules, typically labeled with a
fluorescent dye, are added to the array
• Any reaction between the probe and the
immobilised protein emits a fluorescent signal
that is read by a laser scanner
• rapid, automated, economical, and highly
sensitive, consuming small quantities of
samples and reagents

5. HISTORY
• antibody microarrays / antibody matrix
• 1983 : Roger Ekins
• DNA microarrays
• Limitations : gene expression
• mRNA don’t express proteins
• protein : functional role in cell response
• post-translational modifications :protein function,
are not visible on DNA microarrays
• 2D gel electrophoresis or chromatography

6. PREPARATION OF PROTEIN ARRAY
• microscope slides, membranes, beads or microtitre plates
• Support : protein => immobilized
• binding ability is retained
• Microscope : glass or silicon
• Nitrocellulose : highest protein binding
• solid surface => coating
 immobilising the protein
 preventing its denaturation
 orienting it in the appropriate direction
 binding sites are accessible
 providing a hydrophilic environment

7. • Immobilising agents
 aluminium or gold
 hydrophilic polymers
 polyacrylamide gels
 treatment with amines, aldehyde or epoxy
• Coatings
 physical vapour deposition (PVD)
 chemical vapour deposition (CVD)
• aqueous environment /buffers
 high percent of glycerol (to lower the freezing point)
 humidity of the manufacturing environment

8. METHODS
• robots/robotic contact printingrobot/potting
• ink-jetting
• Piezoelectric spotting
• Photolithography

9. TYPES
• Analytical microarrays/capture arrays
• Functional protein microarrays : target protein
arrays
• Reverse phase protein microarray

11. DETECTION

12. DETECTION

13. APPLICATIONS
• Diagnostics
detection of antigens and antibodies
profiling of sera to discover new
disease biomarkers
monitoring of disease states
responses to therapy
monitoring of environment and food
Digital bioassay
• Proteomics
protein expression profiling

16. • protein functional analysis
identification of protein–protein interactions
protein–phospholipid interactions
small molecule targets
enzymatic substrates
receptor ligands
• antibody characterization
cross-reactivity
Specificity
mapping epitopes
• treatment development
antigen-specific therapies for autoimmunity,
cancer and allergies

17. CHALLANGES
• quite difficult to handle
• Requires more steps than DNA
• Surface & method of attachment
• Producing of array with long shelf life
• Quantifying level of bound protein
• extracting the detected protein from the chip
• reducing non-specific binding by the capture
agents
• capacity of the chip

18. THANKYOU