Coated Slides
Protein MicroArray Program
800 Research Parkway
Meriden, CT 06450 U.S.A.
Tel: (203) 639-2598
Toll Free: (800) 323-1891
Customer Support: (800) 445-7426
Fax: (617) 426-3908
Web site: http://www.perkinelmer.com
Email: hydrogel@packardbioscience.com
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For all other international representatives, contact the
PerkinElmer Life Sciences U.S.A. office.
a 3-dimensional substrate for protein
microarray applications
(for research use only)
NOT FOR RESALE
Protocol Guide
www.perkinelmer.com
Thank you for choosing HydroGelTM coated slides for
your microarray needs!
Nomenclature
In the microarray literature there exist at least two nomenclature
systems for referring to binding partners. Both use common terms:
“probes” and “targets”. We use the following terminology.
Table of Contents
“Probe” is the biological sample fixed to the solid support. For protein
arrays this refers to proteins that are regularly arranged on a solid
substrate and can bind to the target proteins to be detected. For
instance, the probe protein may be selected from the group
consisting of antigens, antibodies, receptors, enzymes, etc., with
antibodies representing a class of the most commonly used probes.
Product Description __________________________ 2
Contents of Package _________________________ 3
Additional Materials and Equipment Required_____ 3
Storage and Handling Prior to Use ______________ 4
“Immobilization” is the process of fixing/binding the probe to the
substrate, in this case the HydroGel coated slide.
Product Use Guidelines _______________________ 4
Preparing HydroGel Coated Slides for Printing _______5
“Target” is the solution phase biological molecule that will bind to the
probe. Targets are usually derived from a biological sample to be
analyzed. “Targets” can refer to a protein, nucleic acid, or other
molecule that is included in samples to be tested and can bind the
probe proteins immobilized on the protein chip.
Printing Buffers and Conditions ___________________6
Immobilization of Proteins on HydroGel Coated Slides 6
Blocking _______________________________________7
Assays and Washes _____________________________7
“Source Plate/Sample Source” is the microtiter plate filled with
probes to be dispensed. Both 96 and 384 well microplates are
commonly used formats.
Imaging________________________________________8
Troubleshooting Guide _______________________ 9
“Array” is an ordered (often orthogonal) set of spots obtained by
dispensing of biological samples (probes) onto a substrate, in this
case, a HydroGel coated slide. One array may contain one or
multiple replicates of the same probe.
Nomenclature ______________________________ 10
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1
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10
Troubleshooting Guide
Symptom
Potential Cause
Damaged, ripped, or
missing substrate
Contact with substrate when in
hydrated form
Large osmotic changes
Diffuse or very large
spots
HydroGel coated slides were
not washed prior to printing
Incompletely dried substrate
prior to printing
Holes in center of
printed spots
Excessive force applied to
substrate by contact printer
Degraded target
Drying during target sample
incubation
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9
15mm
13mm
12 X 40 HydroGel™ Coated Slide
22.2mm
40mm
12mm
Labeled protein level too high
23mm
t
High Background
12mm
ABC
AAA0
0000
Insufficient mixing during
target incubation
Check efficiency of target
labeling
Check quality of sample
preparation (via gel
electrophoresis for example)
Agitate slides on a rocking
platform shaker during target
sample incubation
Use a more dilute target
sample; include a pre-blocking
step; extend washing time
Place slides in a humidified
environment to reduce
evaporation from under cover
glasses
12mm
Insufficient target incubation
time
Poorly labeled target
12 X 12 HydroGel™ Coated Slide
ABC
Low probe immobilization
PerkinElmer HydroGelTM coated slides provide a 3-dimensional
substrate for a variety of protein microarray applications. The
specialized formulation of the HydroGelTM substrate provides a
combination of low inherent fluorescence, low non-specific binding,
and high probe loading capacity while maintaining protein
functionality and accessibility. This unique combination of physical
and chemical properties allows for the development of sensitive,
reproducible assays having a broad dynamic range. HydroGel
coated slides are manufactured and packaged in our Class 1000
clean room to minimize dust and other debris that can interfere with
assays.
t
Weak assay signal
Avoid contact with the
HydroGel substrate
Avoid switching HydroGel
coated slides between high
and low osmotic strength
solutions
Carefully follow the procedure
listed in “Preparing HydroGel
coated slides for printing”
Carefully follow the procedure
listed in “Preparing HydroGel
coated slides for printing”
Optimize printer overtravel
and/or substrate thickness
settings
Do not rinse storage agent
from HydroGel coated slides
until immediately before use
Extend immobilization time;
carry out immobilization in a
chamber containing a dish of
saturated sodium chloride
solution; test alternate printing
buffers
Extend target incubation time
AAA00
000
Storage of unprinted slides
after water rinses
Product Description
Solution
12.8mm
HydroGel coated slides are dried prior to printing. In this state, the
HydroGel substrate supports both non-contact and contact printing.
Contact printers may require an initial adjustment of the over-travel
or substrate thickness settings to produce high quality arrays.
Guidelines for this process are included on a separate sheet.
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2
Immobilization of proteins within the HydroGel substrate requires no
protein modifications. Proteins are irreversibly immobilized within the
porous HydroGel substrate through interactions with the matrix. The
3–dimensional nature of the substrate is thought to minimize protein
denaturation during this process.
The concentration of target, the sample/reaction volume needed for
the assay, the assay temperature and the incubation times are assay
dependent. In general, good performance on HydroGel coated slides
is achieved using sample incubation times from 30 minutes to two
hours. Depending on the size of the target, its concentration, and the
nature of the probes, incubation steps may need to be extended (up
to overnight is possible).
Contents of Package
25 or 100 HydroGel coated slides
HydroGel Coated Slide Protocol Guide
Contact Printer Adjustment Guide
Additional Materials and Equipment Required
Printing buffer options:
0.1M sodium phosphate buffer (pH 7.2)
1X PBS [0.01M sodium phosphate (pH 7.4), 0.14M sodium chloride,
0.003M potassium chloride]
0.05M borate buffer (pH 9.0)
Other:
PBST (1X PBS, 0.5% Tween 20)- sample dilution and wash buffer
Bovine Serum Albumin- blocking agent
(Optional) Fluorescently labeled protein- indicator for printer
adjustment
Equipment:
Printer- for example:
PerkinElmer BioChip Arrayer (non-contact)
or PerkinElmer SpotArrayTM 24 (contact)
Scanner- for example: PerkinElmer ScanArray series confocal
laser scanner
Incubator/oven
Platform Shaker
For dilution of samples, we recommend the use of PBST, however
both the sample dilution buffer and the wash buffer can be varied
based on the assay requirements. Samples containing a purified
target should be supplemented with 1% BSA. If other buffering
systems are used, rapid changes in osmotic strength should be
avoided, as this may lead to substrate damage.
Agitation of the HydroGel coated slides on a rocking platform shaker
during incubations yields optimal performance and sensitivity.
Owing to the 3-dimensional nature of the HydroGel substrate, slightly
longer washes may be required than those typically used on 2dimensional substrates. Post-target incubation wash conditions are
as follows:

Rinse briefly (for 20 seconds) in PBST (PBS + 0.5% Tween
20), followed by three 10 minute washes in fresh changes of
PBST with gentle agitation. Depending on the sample and
detection method, the washes may need to be extended to as
long as 20 minutes each (e.g. when using a directly labeled
complex protein sample containing a high fluorophore
concentration). Slides exposed to different target samples should
be processed separately.
Imaging
HydroGel coated slides can be imaged in any standard microarray
slide scanner, for example PerkinElmer ScanArray. HydroGel
coated slides should be imaged dry.
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3
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8
Blocking
Storage and Handling Prior to Use
Although the HydroGel substrate inherently has very low associated
non-specific binding, blocking has been shown to improve the
resolution of low-abundance targets and consequently may improve
the detection limit of some assays. We recommend that a preblocking step be included prior to the addition of target.
HydroGel coated slides are stable for many months when stored at
room temperature inside the sealed shipper. Refrigeration or
desiccation of HydroGel coated slides is not recommended. After
opening, unused slides should be placed in the original slide box and
the slide box returned to the resealable shipper. Do not store used
HydroGel coated slides with unused HydroGel coated slides.
1. Block in PBST + 1%BSA for one hour at room temp with
gentle agitation.
2. Briefly rinse three times with PBST.
The preprinting wash procedure described below is designed to
remove a storage agent present in the HydroGel substrate. After
washing, do not return unprinted HydroGel coated slides to dry
storage, as they may be susceptible to damage during the printing
and probe immobilization processes. Note that with two-pad
HydroGel coated slides, both pads must be used immediately
following pre-print washing. One pad cannot be used while the other
is “stored” as this can result in damage to the second substrate pad.
Assays and Washes
There are several methods for setting up assays/hybridizations on
HydroGel coated slides. The most convenient way is by use of
disposable gasket-style hybridization chambers. Grace BioLabs sells
HybriWellsTM (catalog numbers HBW75 or HBW2240;
www.gracebio.com) that fit the single pad HydroGel coated slides.
MJ Research sells Frame-Seal™ incubation chambers (catalog
numbers SLF-0601; http://www.mjresearch.com/) that fit the two-pad
HydroGel coated slides. When using these chambers, dry the
HydroGel coated slides as described above, apply the adhesive
gaskets and then the appropriate amount of sample depending on
which gaskets are being used (detailed directions for use are
supplied by the manufacturer). Alternately, approximately 50 to 100
μL of sample can be applied to the array, followed by sealing with a
22 X 50 mm cover glass. When using the cover glass method, the
slides should be placed in a humidified chamber to prevent
evaporation.
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HydroGel coated slides should be used in a dust free, clean
environment and care should be taken to avoid contact with the
coated portion of the slides. Do not use colored ink for labeling
HydroGel coated slides as this may contribute to background
fluorescence.
Product Use Guidelines
The pre-printing and probe immobilization protocols below should be
followed carefully to ensure optimal performance of the HydroGel
substrate. HydroGel coated slides support a number of different
protein microarray applications. Therefore, assay protocols are
largely assay dependent and need to be determined by the
investigator. In general, assay protocols designed for use on 2dimensional substrates can be used on HydroGel coated slides with
some minor modifications; these are discussed below.
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Printing Buffers and Conditions
Preparing HydroGel Coated Slides for Printing
The purpose of the following pre-printing steps is to remove a
storage agent present in the HydroGel substrate and to ensure a
consistent, uniform substrate condition prior to printing.
The HydroGel substrate is compatible with phosphate and borate
buffer systems and glycerol concentrations up to 40%. The HydroGel
substrate is stable from pH 5 to 9. Protein immobilization is affected
by pH.
1. Briefly rinse HydroGel slides three times in distilled water
(dH2O), followed by three washes in dH2O for 10 minutes
each, and finally six brief rinses in dH2O. All wash steps are
most effective when carried out in a large volume, for example,
by grouping the slides in a 10 slide-capacity glass slide dish and
washing with a 200mL volume. Always use fresh changes of
dH2O for each wash step.
2. Remove excess/free water. Excess water can be removed by
spinning the HydroGel coated slides gently in a benchtop
centrifuge (at approximately 1000 to 1500 RCF for 5 minutes).
Caution: when hydrated, the HydroGel substrate is not rigid and
centrifuging at higher speeds or for longer times may damage
the coating. Alternately, HydroGel coated slides can be dried by
placing them vertically on top of an absorbent surface for several
minutes to allow excess water to run off the slides. Caution: do
not place HydroGel coated slides substrate side down or directly
blot the HydroGel substrate, as this will cause damage. When
dry, the HydroGel substrate has a clear, smooth and thin
appearance.
3. Place HydroGel slides in a 40o C incubator/oven for 20
minutes. Caution: printing on wet slides can sometimes lead to
substrate damage and undesirably large, poorly shaped spots.
4. Remove from incubator/oven and allow slides to cool to
room temperature (approximately 5 minutes).
To avoid edge effects, do not print within 1 mm from the edges of the
HydroGel substrate.
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Immobilization of Proteins on HydroGel Coated
Slides
The immobilization of proteins within the HydroGel substrate
correlates directly with post-printing incubation time and is
dependent on both the printing buffer and the proteins themselves.
1. Incubate arrays in a humidified chamber. Efficient
immobilization requires an extended post-printing incubation (8
to 16 hours). For thermally stable proteins the incubation should
be conducted at 30C. Placing a saturated sodium chloride
solution at the bottom of a standard incubator at 30 C yields the
proper level of relative humidity (approximately 65%). When
using a particularly fragile, thermal sensitive, or impure protein
the incubation can be carried out at a lower temperature, such as
4C. Note however that the incubation time will have to be
extended when the incubation is carried out at a lower
temperature. If data are to be quantitatively compared among
multiple days, this incubation time should be held constant.
2. Rinse briefly (for 20 seconds) in PBST (PBS + 0.5% Tween
20), followed by three 30 minute washes with PBST.
3. (Optional) Dry HydroGel coated slides. After printing and
immobilization, slides can be stored in blocking solution (with the
addition of 0.01% sodium azide) or dry at 4C (non-condensing
atmosphere). No significant loss of probe activity (either IgG or
streptavidin probes) was found for up to one week of storage in
either condition. Since the stability of printed probes is protein
dependent, the user is encouraged to evaluate the storage
periods for the specific assay.
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