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Protein Microarrays: Approaches to Printing
1.
2. 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..
3. Protein MicroarraysProtein Microarrays
• Immobilize proteins onto small
surfaces (in large numbers)
• Using proven and existing technology –
DNA microarrays
• Specialized protein technology
6. Protein Microarray WorkflowProtein Microarray Workflow
• Important factors:
• Printing – Focus of this talk
• Surface -Substrate
• Imaging
7.
8. 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
9. Protein MicroarraysProtein Microarrays
• Substrates:
• 3 dimensional
• Specialized for protein applications:
• Slide H - hydrogel (Schott Nexterion)
• FAST - nitrocellulose (Whatman)
• Slide format:
10. Printing onto 3D substratesPrinting onto 3D substrates
Microgrid II Omnigrid Accent
Contact printing/split pins
11. Considerations for 3D surfacesConsiderations for 3D surfaces
• Avoid Damage to 3D surface –
12. 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).
13. 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
14. Considerations for 3D surfacesConsiderations for 3D surfaces
• Avoid Damage to 3D surface –
15. 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
16. 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
17. 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
18. 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:
19. 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
23. 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.
25. 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
27. 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
28. 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
37. 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
43. 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
47. 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
Notes de l'éditeur
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