protein microarrays: approaches to printing
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Protein Microarrays:Protein Microarrays:Approaches to PrintingApproaches to Printing
Steven Suchyta Ph. D.Steven Suchyta Ph. D.Applications ScientistApplications ScientistGenomic Solutions IncGenomic Solutions Inc..
Protein MicroarraysProtein Microarrays
• Immobilize proteins onto small surfaces (in large numbers)
• Using proven and existing technology – DNA microarrays
• Specialized protein technology
Protein MicroarraysProtein Microarrays
• Protein Microarray Workflow• Measure protein expression • Antibody binding• Screen & assess patterns of
interaction
Significance:Significance:
>50 citations in Genbank in 2005
Protein Microarray WorkflowProtein Microarray Workflow
• Important factors:• Printing – Focus of this talk• Surface -Substrate• Imaging
Optimizing protein microarray Optimizing protein microarray spottingspotting
• For visualizing spots for optimization – do not want to use costly protein.
• Red food dye (~5%) in the spotting buffer
• Fluoresces in the CY3 channel
Protein MicroarraysProtein Microarrays• Substrates:• 3 dimensional • Specialized for protein
applications:• Slide H - hydrogel (Schott
Nexterion)• FAST - nitrocellulose (Whatman) • Slide format:
Printing onto 3D substratesPrinting onto 3D substrates
Microgrid II Omnigrid Accent
Contact printing/split pins
Considerations for 3D surfacesConsiderations for 3D surfaces
• Avoid Damage to 3D surface –
Omnigrid System:
Contact of the pin to the surface can be precisely calibrated.
Z Coordinate of the pin tip as it touches the surface can be finely adjusted (2.5 micron resolution).
Microgrid System:
Soft Touch: greatly reduce the speed of travel of the print head before it reaches the slide surface.
100mm/s to ~1 – 4 mm/s
Considerations for 3D surfacesConsiderations for 3D surfaces
• Avoid Damage to 3D surface –
Minimizing Carry-over Minimizing Carry-over ContaminationContamination
• Composition and differing viscosity of various spotting solutions – PBS, Glycerol
• Minimize the amount of carry-over between samples
Minimizing Carry-over Minimizing Carry-over ContaminationContamination
Omnigrid Accent
•Complete control over the wash steps (time)
•Cycles through the wash protocol•Wash
•Sonication station
•Vacuum dry
Minimizing Carry-over Minimizing Carry-over ContaminationContamination
Microgrid II
• 2 recirculating wash baths – can be stationary (etoh)
• Main Wash station “flood and flush”
• vacuum dry
Complete control over the wash steps (time)Cycles through the wash protocol
Minimizing Carry-over Minimizing Carry-over ContaminationContamination
• 2 systems have different wash setups • BOTH were shown to be equally effective for
cleaning pins.
• Empirically determine proper wash protocol:
Protein microarray Experiment –Protein microarray Experiment –3D substrate3D substrate
• Print 5mg/ml of rabbit IgG (Invitrogen)
• 3X3 array onto the pads of a 8 Pad FAST slide (Whatman)
• Solution phase binder - 100ug/ml of FluoroLink Cy3-labeled goat anti-rabbit IgG (GE Healthcare)
Genetic Engineering News – Assay Tutorial – Jan. 2006
Binding AssayBinding Assay
• Genomic Solutions HybStation
• Binding • Washing
Protein microarray Experiment –Protein microarray Experiment –3D substrate3D substrate
Protein MicroarraysProtein Microarrays
• Traditional DNA chemistry for protein applications: Epoxy, aldehyde
• Slide E, AL (Schott Nexterion)
• Multiplexed Assay: Microarray assembled in order to assay a large number (16, 96, 384) of samples in parallel to decrease cost and increase assay reproducibility.
Multiplexed Protein Multiplexed Protein MicroarraysMicroarrays
Slide MPX
Plate MTP
Plate can be printed separately or inside a tray
Maximum printing area is available (up to 2000 probes/well)
Analysis of 96 targets simultaneously
Design conforms to the SBS standards and suited for high throughput robotic handling
Designed to eliminate cross-contamination
Chemistries optimized for DNA and protein applications
Nexterion MTP has a Unique Nexterion MTP has a Unique DesignDesign
Ultra hydrophobic pattern ensures negligible cross-contamination across wells of multiplexed substrates
2082 26971167
7331 8575 5483
532 592348
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Gene 51 Gene1765
Gene1833
Gene 51 Gene1765
Gene1833
Slides/Conditions
To
tal M
ed
ian
Sig
na
l
Target No Target
non-MPX MPX
1 2
3 4
5 6
7 8
1 2
3 4
5 6
7 8
Partitioned
1 2
3 4
5 6
7 8
1 2
3 4
5 6
7 8
1 2
3 4
5 6
7 8
1 2
3 4
5 6
7 8
Target added to wells 1, 4, 5, and 8 only
Simple target Complex TargetNon-partitioned PartitionedNon-partitioned
<3% contamination in partitioned wells
Contamination not detectable in partitioned wells
100
50
75
25
0
Cy3 Cy5
Nor
mal
ized
sig
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Afte
r hy
brid
izat
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Target volume: 60 µl 45 µl 30 µl Target amount: 7.5 pmol 7.5 pmol 7.5 pmol
166 nM
125 nM
250 nM
Different volumes can be used for hybridization Increased sensitivity can be achieved using lower volumes
Increased Sensitivity on Multiplexed Increased Sensitivity on Multiplexed SubstratesSubstrates
Protein Array printing on MTP Protein Array printing on MTP platesplates
• In addition to good spot morphology
• No carry over between samples
• Axis Stability - Stable positioning into EACH well of a multiwell plate to consistently position array in relation to well wall: Microgrid II, Omnigrid Accent
• Easy to use software
Microgrid Plate printingMicrogrid Plate printing
Easy to use plate printing interface: PlateArray
Wizard
Multiplex Plate Arraying on MicroGrid IIMultiplex Plate Arraying on MicroGrid II
• Capacity– 24 source plates– 16 target plates
• Source plate cooling• Plate & lid handling for
sample plates• Humidity controlled• HEPA filtered• “Soft-touch” software• Re-circulating wash baths
& high pressure pin drying• Barcode reading option
Omnigrid Accent plate printing
• Software upgrade:
Medium throughput: 6 target plates, 3 source plates at a time. Humidity, plate cooling
Multiplex Protein MicroArrayMultiplex Protein MicroArray
• Substrate: MTP 96 (Slide E or AL)
• Printer: Micrigrid II/Omnigrid Accent
• Imaging: Alpha Innotech - Novaray
• Alpha Innotech’s NovaRay ® Alpha Innotech’s NovaRay ® brings the multiplexing of brings the multiplexing of arrays to high throughput arrays to high throughput screening.screening.
• Multi-format testing capabilities– Microplates– Slides– Custom formats
• Multispectral imaging– Broad-band light source– 8 emission wavelengths
NovaRay ®: A Novel High NovaRay ®: A Novel High Throughput Multiplexed Array Throughput Multiplexed Array Detection PlatformDetection Platform
Gene Arrays
Protein Arrays
Cell Arrays
Chemical Compound Arrays
Primary Focus
Secondary Focus
NovaRay ® Supports Multiple ApplicationsNovaRay ® Supports Multiple Applications
Compatible dyes Excitation Wavelength Emission Wavelength
Cy3, Cy3.5, Alexa 546, TRITC, JOE, ROX, TAMRA 480-552 572-640
Cy3 527-552 570-620
Cy5, Cy5.5, Alexa 647, Alexa 680, Bodipy 630/650 572-640 664-750
Cy5 620-650 665-705
FITC, Alexa 488, FAM, PicoGreen, GFP 455-495 518-568
Texas Red 540-597 615-665
NIR 675-725 730-830
Phycoerythrin 480-552 664-750
NovaRay ® Supported WavelengthsNovaRay ® Supported Wavelengths
1
3
5
7
Similar images obtained in different wells on a multiplexed substrate
2
3
6
4
8
Average Log Intensity (ALI) = [Log10(Cy3+Cy5)/2]
Data from individual wells can be quantitativelycompared using regression analysis
0.5
1.0
1.5
2.0
2.5
1.0 1.5 2.0 2.5
R2=0.98
0.5
1.0
1.5
2.0
2.5
1.0 1.5 2.0 2.5ALI (Subarray 3)
ALI
(Sub
arra
y5)
R2=0.98
Average R2 = 0.96 ± 0.03
1 2 3 4 5 6 7 81 0.96 0.93 0.93 0.91 0.93 0.91 0.932 0.96 0.95 0.95 0.93 0.95 0.94 0.953 0.93 0.95 0.98 0.98 0.98 0.98 0.994 0.93 0.95 0.98 0.98 0.98 0.98 0.995 0.91 0.93 0.98 0.98 0.97 0.99 0.986 0.93 0.95 0.98 0.98 0.97 0.98 0.987 0.91 0.94 0.98 0.98 0.99 0.98 0.988 0.93 0.95 0.99 0.99 0.98 0.98 0.98
1 2 3 4 5 6 7 81 0.96 0.93 0.93 0.91 0.93 0.91 0.932 0.96 0.95 0.95 0.93 0.95 0.94 0.953 0.93 0.95 0.98 0.98 0.98 0.98 0.994 0.93 0.95 0.98 0.98 0.98 0.98 0.995 0.91 0.93 0.98 0.98 0.97 0.99 0.986 0.93 0.95 0.98 0.98 0.97 0.98 0.987 0.91 0.94 0.98 0.98 0.99 0.98 0.988 0.93 0.95 0.99 0.99 0.98 0.98 0.98
Subarray
A pair-wise comparison can be used to assessoverall reproducibility
Excellent Reproducibility (Excellent Reproducibility (R2> 0.9 ) on Multiplexed ) on Multiplexed SubstratesSubstrates
Signal vs Exposure Time
R2 = 0.9938
0
10000
20000
30000
40000
50000
60000
500 ms 1000 ms 2000 ms 3000 ms 4000 ms 5000 ms
Expsoure time (milliseconds)
Rel
ativ
e In
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Val
ue
• Multispot array target.
• Linear imaging results with increase in exposure time.
•High correlation between increasing exposure time (R2 = 0.9938)
Linear Response: User ValidationLinear Response: User Validation
Integrated Muliplex PlatformIntegrated Muliplex Platform
Results !Results !
• Cell based time-point studies
• Lead optimization• Clinical trials• Biomarker screening and
validation• Cell Based Assays• Enzyme Assays • Functional Assays • In Vitro Toxicology • Biomarker screening and
validation• Antiviral Assays• Novel Targets Panel
Multiplex Array ApplicationsMultiplex Array Applications
Integrated Multiplex PlatformIntegrated Multiplex PlatformProtein MicroarrayProtein Microarray
• Print 0.1 mg/ml rabbit IgG onto Nexterion Slide E MTP 96 Epoxy substrate • 3X3 subarray in each well – Microgrid II
• Solution phase binder - 10ug/ml of FluoroLink Cy3-labeled goat anti-rabbit IgG (GE Healthcare) (Schott Nexterion protocol)
• Scan image with the Alpha Innotech Novaray
Results !Results !
Integrated Multiplex PlatformIntegrated Multiplex PlatformProtein MicroarrayProtein Microarray
Cy3 labeled
anti-rabbit IgG bound to rabbit
IgG
Integrated Multiplex PlatformIntegrated Multiplex PlatformProtein MicroarrayProtein Microarray
Anti-rabbit IgG Negative
Anti-IgG
Anti-HSA
Probes detected
All IgG HSA
IgG and HSA proteins are specifically detected by their respective antibodies
MPX
MTP
No carry over
Anti-STP
Integrated Multiplex PlatformIntegrated Multiplex PlatformProtein MicroarrayProtein Microarray
SummarySummary
• SCHOTT: MPX slide and plate formats
• Simultaneous biological sample analysis• Versatile assay design possibilities• Critical for diagnostic/applied research
• Genomic Solutions: MicroGrid II & OmniGrid Accent
• OmniGrid Accent medium throughput plate arraying• MicroGrid II high throughput• Available to new & existing customers
• Alpha Innotech: NovaRay® Plate reader• Supports multiple wavelength assays• High resolution capability• Multi-chamber slide and 96 well plate imaging
Protein Microarray WorkflowProtein Microarray Workflow
AcknowledgementsAcknowledgements
• Genomic Solutions
– Dr. Jeremy Clarke
– Dr. Sally Johnston
– Dr. Jennifer Owen
– Dr. Jim Galt
– Dr. Hamid Khoja
– Judy Thompson
• Schott Nexterion
– Dr. Rajendra Redkar
– Dr. Luis Burzio
– Heather Jakes
• Alpha Innotech
Dr. Mike Collier