Protein Microarrays:Protein Microarrays:
Approaches to PrintingApproaches to Printing
Steven Suchyta Ph. D.Steven Suchyta ...
Protein MicroarraysProtein Microarrays
• Immobilize proteins onto small
surfaces (in large numbers)
• Using proven and exi...
Protein MicroarraysProtein Microarrays
• Protein Microarray Workflow
• Measure protein expression
• Antibody binding
• Scr...
Significance:Significance:
>50 citations in Genbank in 2005
Protein Microarray WorkflowProtein Microarray Workflow
• Important factors:
• Printing – Focus of this talk
• Surface -Sub...
Optimizing protein microarray spottingOptimizing protein microarray spotting
• For visualizing spots for optimization – do...
Protein MicroarraysProtein Microarrays
• Substrates:
• 3 dimensional
• Specialized for protein applications:
• Slide H - h...
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 ...
Microgrid System:
Soft Touch: greatly
reduce the speed of
travel of the print
head before it
reaches the slide
surface.
10...
Considerations for 3D surfacesConsiderations for 3D surfaces
• Avoid Damage to 3D surface –
Minimizing Carry-over ContaminationMinimizing Carry-over Contamination
• Composition and differing viscosity of
various sp...
Minimizing Carry-over ContaminationMinimizing Carry-over Contamination
Omnigrid Accent
•Complete control over the wash ste...
Minimizing Carry-over ContaminationMinimizing Carry-over Contamination
Microgrid II
• 2 recirculating wash baths – can be
...
Minimizing Carry-over ContaminationMinimizing Carry-over Contamination
• 2 systems have different wash setups
• BOTH were ...
Protein microarray Experiment –Protein microarray Experiment –
3D substrate3D substrate
• Print 5mg/ml of rabbit
IgG (Invi...
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...
Multiplexed Protein MicroarraysMultiplexed Protein Microarrays
Slide MPX
Plate MTP
 Plate can be printed separately or inside a tray
 Maximum printing area is available (up to 2000 probes/well)
 Analysi...
Ultra hydrophobic pattern ensures negligible cross-
contamination across wells of multiplexed substrates
2082 2697
1167
73...
100
50
75
25
0
Cy3 Cy5
Normalizedsignal
Afterhybridization
Target volume: 60 µl 45 µl 30 µl
Target amount: 7.5 pmol 7.5 pm...
Protein Array printing on MTP platesProtein Array printing on MTP plates
• In addition to good spot
morphology
• No carry ...
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...
Omnigrid Accent plate printing
• Software upgrade:
Medium throughput: 6 target plates, 3 source
plates at a time. Humidity...
Multiplex Protein MicroArrayMultiplex Protein MicroArray
• Substrate: MTP 96 (Slide E or AL)
• Printer: Micrigrid II/Omnig...
• Alpha Innotech’s NovaRay ®Alpha Innotech’s NovaRay ®
brings the multiplexing ofbrings the multiplexing of
arrays to high...
Gene Arrays
Protein Arrays
Cell Arrays
Chemical Compound
Arrays
Primary Focus
Secondary Focus
NovaRay ® Supports Multiple ...
Compatible dyes Excitation Wavelength Emission Wavelength
Cy3, Cy3.5, Alexa 546, TRITC,
JOE, ROX, TAMRA 480-552 572-640
Cy...
1
3
5
7
Similar images obtained in different
wells on a multiplexed substrate
2
3
6
4
8
Average Log Intensity (ALI) = [Log...
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
E...
Integrated Muliplex PlatformIntegrated Muliplex Platform
Results !Results !
• Cell based time-point studies
• Lead optimization
• Clinical trials
• Biomarker screening and
validation
• Cell Based As...
Integrated Multiplex PlatformIntegrated Multiplex Platform
Protein MicroarrayProtein Microarray
• Print 0.1 mg/ml rabbit I...
Integrated Multiplex PlatformIntegrated Multiplex Platform
Protein MicroarrayProtein Microarray
Cy3
labeled
anti-rabbit
Ig...
Integrated Multiplex PlatformIntegrated Multiplex Platform
Protein MicroarrayProtein Microarray
Anti-rabbit IgG Negative
A...
Integrated Multiplex PlatformIntegrated Multiplex Platform
Protein MicroarrayProtein Microarray
SummarySummary
• SCHOTT: MPX slide and plate formats
• Simultaneous biological sample analysis
• Versatile assay design po...
Protein Microarray WorkflowProtein Microarray Workflow
AcknowledgementsAcknowledgements
• Genomic Solutions
– Dr. Jeremy Clarke
– Dr. Sally Johnston
– Dr. Jennifer Owen
– Dr. Ji...
Protein Microarrays: Approaches to Printing
Protein Microarrays: Approaches to Printing
Protein Microarrays: Approaches to Printing
Protein Microarrays: Approaches to Printing
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Protein Microarrays: Approaches to Printing

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  • Using proven and existing technology as well as specialized technology designed specifically for protein arrays.
  • Diagram: shows a typical protein array experiment: proteins being printed, assay, and imaging.
  • From Genbank search microarray AND protein in title
  • 3 important factors – this has brought the 3 companies together. Printing (this talk) Genomic Solutions, Substrates and surfaces (Schott), and Imaging (AlphaInnotech)
  • Quick note about optimizing protein microarrays
  • One choice of substrate – 3 dimensional
    Talk about the Slide H: see below.
    For FAST: using nitrocellulose like a Western blot.
    Some FAST slides have individual “pads” of nitrocellulose
    Nexterion® Slide H is ideally suited for covalent immobilization of peptides and proteins such as antibodies, antibody fragments, enzymes, or receptors.
    The permeable, 3D hydrogel coating preserves the native three-dimensional structure of proteins thereby maintaining protein stability and functionality.
    Nexterion® Slide H enables excellent signal-to-background ratios and exceptionally high linear dynamic ranges compared to conventional 2D coatings by a unique combination of low non-specific binding characteristics, high loading capacity, and the use of glass with low auto-fluorescence. Even very low intensity signals, such as those from low-abundance proteins or weakly expressed genes can be reliably detected and quantified. The density of the reactive groups is consistent over the entire surface and is optimized to maximize binding capacity. The reactive chemistry is stable and remains active even during very long spotting runs.
  • Want to use traditional proven DNA microarray instrumentation – such as our high-throughput Micgrid (120 slides)
    Or the Omnigrid Accent (50 slides) medium throughput. No changes – still use contact printing with split pins.
  • Exaggerated image of a split pin hitting the surface of a slide – want to avoid the image on the left
    For these machines this is done via the software.
  • Adjust so there is deposit of protein, but no damage to the surface.
  • You do calibrate the point at which the pins touch (Target Height). In addition has Soft Touch.
    When slowed down – reduce/eliminate damage to the surface.
  • When the contact of the split pin is optimized can eliminate 3d surface damage – image on the left
  • Due the composition and viscosity – it can be a challenge to try and minimize or eliminate carry over between samples
  • Have complete control over the was steps – Omngrid has an onboard sonicator.
    Can empirically determine what wash steps and cycles work best
  • Also have complete control over the wash cycles/steps. Can use etoh in stationary baths
  • Experiment: print 6 spots (red food dye in 1X PBS), wash, print 6 spots in 1X PBS (no dye) (Cy3 channel)
    Image on left shows carry over from column 3 tp 4
  • Gone through the optimization – real experiment –
    Allowed to bind 1 hours at RT (resuspended in PBST), washed with PBST
  • Using DNA microarray equipment to do the assay
  • Scanned on cy3 channel – conversion of a Western blot to a microarray format
    Want to show that a western type assay can be converted to a micro array format
  • Switch gears – a lot of the first stuff remains true – optimization, wash cycles etc…
    Get away from 3d substrates to more traditional – epoxy etc…
    Also – multiplexed Assay
  • Allows you to do a large number of samples in parallel on the same slide or in a plate
    Slide – can use automation set up for slides – printer
    Plate – can use automation for plates – plate handling, liquid handling, plate readers, plus a large number of samples in parallel
  • Going to focus on the plate mutliplex assay
  • Start on technical aspects of MTP (2 slides)
  • We decided on what type of plate type – MTP
    Now what to use for printing -
    No damage, be able to wash, previous part
    Also mechanical ability, software use
  • Change from slide holder to plate holder on robot
    Choose what type of plate, subarray pattern, how many plates - replicates
  • Software allows you pick how many spots in each well, replicates, and which wells to use - replicates
  • Have decided on using MTP, and GSI printer
    But traditional microarray (slide) scanner will not work
    Use the Novaray
  • Technical aspects (3 slides)
  • Uniformity of image over the surface of the slide
    Over multiple wells – important for plate arrays
  • MTP/MPX, GSI arrayers, Novaray,
    Integrated Mulitplex protein microarray platform
  • What kind of results -
  • Demonstration of the integrated multiplexed protein microarray platform – antibody binding assay
    Important – can print a lot less when not using 3D substrate before 5mg/ml – 50 fold less, 100ug/ml target – 10 fold less
    1 hour at room temperature following Schott Nexterion protocol – done on lab bench
  • The last three columns of the plate
  • Left – both printed with IgG – left target , right only PBST
    Left done on MTP
    Right – earlier work done on the MPX 16 slides
  • Lastly, high quality images generated –
    Good spot morphology, low background,
  • GSI, Schott, and Alpha come together to simplify the protein microarray workflow
  • Protein Microarrays: Approaches to Printing

    1. 1. Protein Microarrays:Protein Microarrays: Approaches to PrintingApproaches to Printing Steven Suchyta Ph. D.Steven Suchyta Ph. D. Applications ScientistApplications Scientist Genomic Solutions IncGenomic Solutions Inc..
    2. 2. Protein MicroarraysProtein Microarrays • Immobilize proteins onto small surfaces (in large numbers) • Using proven and existing technology – DNA microarrays • Specialized protein technology
    3. 3. Protein MicroarraysProtein Microarrays • Protein Microarray Workflow • Measure protein expression • Antibody binding • Screen & assess patterns of interaction
    4. 4. Significance:Significance: >50 citations in Genbank in 2005
    5. 5. Protein Microarray WorkflowProtein Microarray Workflow • Important factors: • Printing – Focus of this talk • Surface -Substrate • Imaging
    6. 6. Optimizing protein microarray spottingOptimizing protein microarray spotting • 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
    7. 7. Protein MicroarraysProtein Microarrays • Substrates: • 3 dimensional • Specialized for protein applications: • Slide H - hydrogel (Schott Nexterion) • FAST - nitrocellulose (Whatman) • Slide format:
    8. 8. Printing onto 3D substratesPrinting onto 3D substrates Microgrid II Omnigrid Accent Contact printing/split pins
    9. 9. Considerations for 3D surfacesConsiderations for 3D surfaces • Avoid Damage to 3D surface –
    10. 10. 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).
    11. 11. 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
    12. 12. Considerations for 3D surfacesConsiderations for 3D surfaces • Avoid Damage to 3D surface –
    13. 13. Minimizing Carry-over ContaminationMinimizing Carry-over Contamination • Composition and differing viscosity of various spotting solutions – PBS, Glycerol • Minimize the amount of carry-over between samples
    14. 14. Minimizing Carry-over ContaminationMinimizing Carry-over Contamination Omnigrid Accent •Complete control over the wash steps (time) •Cycles through the wash protocol •Wash •Sonication station •Vacuum dry
    15. 15. Minimizing Carry-over ContaminationMinimizing Carry-over Contamination 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
    16. 16. Minimizing Carry-over ContaminationMinimizing Carry-over Contamination • 2 systems have different wash setups • BOTH were shown to be equally effective for cleaning pins. • Empirically determine proper wash protocol:
    17. 17. 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
    18. 18. Binding AssayBinding Assay • Genomic Solutions HybStation • Binding • Washing
    19. 19. Protein microarray Experiment –Protein microarray Experiment – 3D substrate3D substrate
    20. 20. 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.
    21. 21. Multiplexed Protein MicroarraysMultiplexed Protein Microarrays Slide MPX Plate MTP
    22. 22.  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 DesignNexterion MTP has a Unique Design
    23. 23. Ultra hydrophobic pattern ensures negligible cross- contamination across wells of multiplexed substrates 2082 2697 1167 7331 8575 5483 532 592 348 2 1 2 1 10 100 1000 10000 100000 Gene 51 Gene 1765 Gene 1833 Gene 51 Gene 1765 Gene 1833 Slides/Conditions TotalMedianSignal 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 Target Non-partitioned PartitionedNon-partitioned  <3% contamination in partitioned wells  Contamination not detectable in partitioned wells
    24. 24. 100 50 75 25 0 Cy3 Cy5 Normalizedsignal Afterhybridization 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 MultiplexedIncreased Sensitivity on Multiplexed SubstratesSubstrates
    25. 25. Protein Array printing on MTP platesProtein Array printing on MTP plates • 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
    26. 26. Microgrid Plate printingMicrogrid Plate printing Easy to use plate printing interface: PlateArray Wizard
    27. 27. 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
    28. 28. Omnigrid Accent plate printing • Software upgrade: Medium throughput: 6 target plates, 3 source plates at a time. Humidity, plate cooling
    29. 29. Multiplex Protein MicroArrayMultiplex Protein MicroArray • Substrate: MTP 96 (Slide E or AL) • Printer: Micrigrid II/Omnigrid Accent • Imaging: Alpha Innotech - Novaray
    30. 30. • Alpha Innotech’s NovaRay ®Alpha Innotech’s NovaRay ® brings the multiplexing ofbrings the multiplexing of arrays to high throughputarrays 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 ThroughputNovaRay ®: A Novel High Throughput Multiplexed Array Detection PlatformMultiplexed Array Detection Platform
    31. 31. Gene Arrays Protein Arrays Cell Arrays Chemical Compound Arrays Primary Focus Secondary Focus NovaRay ® Supports Multiple ApplicationsNovaRay ® Supports Multiple Applications
    32. 32. 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
    33. 33. 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 quantitatively compared 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 2.5 ALI (Subarray 3) ALI(Subarray5) R2 =0.98 Average R2 = 0.96 ± 0.03 1 2 3 4 5 6 7 8 1 0.96 0.93 0.93 0.91 0.93 0.91 0.93 2 0.96 0.95 0.95 0.93 0.95 0.94 0.95 3 0.93 0.95 0.98 0.98 0.98 0.98 0.99 4 0.93 0.95 0.98 0.98 0.98 0.98 0.99 5 0.91 0.93 0.98 0.98 0.97 0.99 0.98 6 0.93 0.95 0.98 0.98 0.97 0.98 0.98 7 0.91 0.94 0.98 0.98 0.99 0.98 0.98 8 0.93 0.95 0.99 0.99 0.98 0.98 0.98 1 2 3 4 5 6 7 8 1 0.96 0.93 0.93 0.91 0.93 0.91 0.93 2 0.96 0.95 0.95 0.93 0.95 0.94 0.95 3 0.93 0.95 0.98 0.98 0.98 0.98 0.99 4 0.93 0.95 0.98 0.98 0.98 0.98 0.99 5 0.91 0.93 0.98 0.98 0.97 0.99 0.98 6 0.93 0.95 0.98 0.98 0.97 0.98 0.98 7 0.91 0.94 0.98 0.98 0.99 0.98 0.98 8 0.93 0.95 0.99 0.99 0.98 0.98 0.98 Subarray A pair-wise comparison can be used to assess overall reproducibility Excellent Reproducibility (Excellent Reproducibility (R2 > 0.9 ) on Multiplexed) on Multiplexed SubstratesSubstrates
    34. 34. 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) RelativeIntensityValue • 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
    35. 35. Integrated Muliplex PlatformIntegrated Muliplex Platform Results !Results !
    36. 36. • 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
    37. 37. Integrated Multiplex PlatformIntegrated Multiplex Platform Protein 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 !
    38. 38. Integrated Multiplex PlatformIntegrated Multiplex Platform Protein MicroarrayProtein Microarray Cy3 labeled anti-rabbit IgG bound to rabbit IgG
    39. 39. Integrated Multiplex PlatformIntegrated Multiplex Platform Protein 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
    40. 40. Integrated Multiplex PlatformIntegrated Multiplex Platform Protein MicroarrayProtein Microarray
    41. 41. 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
    42. 42. Protein Microarray WorkflowProtein Microarray Workflow
    43. 43. 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
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