Arrayit
Polypropylene Rinse Buffer (Cat. PRB) rinses polypropylene slides, substrates
and other surfaces after functionalization in preparation for protein binding
and microarray assays. Polypropylene Rinse Buffer is used in conjunction with
Arrayit Polypropylene Functionalization Buffer (Cat. PFB) to enhance binding of
antibodies, antigens, peptides, recombinant proteins, cell extracts and other
proteins to polypropylene surfaces for immunoassays and other applications.
Arrayit PRB is supplied as 1 liter of 1X solution and is ready to use.
Reagents
- Microarray Buffers - Polypropylene Rinse Buffer to Enhance Polypropylene
Microarray Assays
Arrayit’s
Polypropylene Rinse Buffer (Cat. PRB) extends protein microarray assays by opening
up the use of polypropylene slides, substrates and other plastic surfaces for
protein microarray assays including immunoassays. Arrayit PRB removes the
guesswork associated with polypropylene-based assays by allowing users to rinse
plastic surfaces after functionalization to remove Functionalization buffer
prior to protein binding. Buffer PRB is pre-mixed and ready to use at a 1X solution.
This buffer is formulated to increase signal strength and reduce background by
promoting high-density protein binding and optimal reactivity. Highly
recommended for peptide, antibody, antigen, reverse phase and other types of
microarrays and immunoassays.
Table of Contents
- Introduction
- Quality Control
- Product Description
- Buffer Contents
- Technical Assistance
- Polypropylene Slide Functionalization Protocols
- Polypropylene Slide Protein Binding Protocols
- Polypropylene Slide Protein-Protein Binding
Protocols
- Troubleshooting Tips
- Recommended Products and Ordering Information
- Warranty
Introduction
Arrayit Polypropylene Rinse Buffer improves the precision, speed and
affordability of your microarray research in proteomics, pharmaceuticals, life
sciences, agriculture, and other areas of biomedical research. This handbook
contains all the information required to take full advantage of Arrayit
Polypropylene Rinse Buffer.
Quality Control
Arrayit uses the highest quality control (QC) and quality assurance (QA)
measures to ensure the quality of our Arrayit brand Polypropylene Rinse Buffer.
The finest scientific research was used to develop this product. The use of
advanced R&D, ultra-pure reagents, and quantitative preparation guarantees
that this product performs to the highest industry standards.
Product Description
Arrayit Polypropylene Rinse Buffer is designed for all types of protein
microarray applications that use plastic slides, substrates and sheets. Users
will appreciate the following features:
- Based on the finest microarray research and
development (R&D)
- Excellent for protein immunoassays and protein
microarray applications
- Designed for proteins, antigens, peptides, cell
extracts and serum samples
- Promotes strong signal intensities and high
binding capacity
- Superior formulations reduce background
- Each lot checked by quality control (QC) and
quality assurance (QA)
- Ultra-pure reagents used for buffer formulation
- All buffers quantitatively prepared
- Provided in convenient 1 liter bottles as a 1X
solution
- Arrives pre-mixed and ready to use
- Remove the guesswork from plastics-based protein
microarrays
Buffer Contents
Arrayit Polypropylene Rinse Buffer contains the following:
- 1 liter (1000 ml) of 1X Polypropylene Rinse
Buffer at a 1X concentration
Technical Assistance
Please contact us if you have any questions, comments, suggestions, or
if you need technical assistance. By electronic mail, use arrayit@arrayit.com and type "ArrayIt technical
assistance" in the subject line for immediate response between 8 AM-8 PM
PST Monday-Friday. We want to hear about your successes and are always happy to
feature contributed data on our website.
Polypropylene Slide Functionalization
Short Protocol
1. Wear white nitrile gloves
and safety glasses at all times during this protocol.
2. Heat Arrayit
Polypropylene Functionalization Buffer (PFB) to 100°C.
3. Incubate polypropylene slides
in 100°C PFB for 2 hours.
4. Rinse polypropylene slide
exhaustively with tap water for 5 minutes.
5. Heat Arrayit
Polypropylene Rinse Buffer (PRB) to 100°C.
6. Rinse polypropylene slides
once with PRB to remove residual tap water.
7. Incubate polypropylene slides
in 100°C PRB for 1 hour.
8. Dry polypropylene slides overnight
at 37°C.
Polypropylene Slide Functionalization
Complete Protocol
1. Wear white nitrile gloves
and safety glasses at all times during this protocol. Nitrile will protect your
hands during this process and prevent the transfer of hand oils and other
contaminants onto the polypropylene surface. Pigmented nitrile (e.g. blue and
black) should be avoided as these pigments can be transferred onto the surface
during handling. Safety glasses will provide eye protection against chemical
splashes and hot liquids.
2. Heat Arrayit
Polypropylene Functionalization Buffer (PFB) to 100°C. Heating can be performed
in a microwave oven or on a hot plate. Make sure to wear nitrile gloves and safety
glasses at all times when handling PFB, and avoid spills onto painted surfaces
as PFB is corrosive. Temperatures lower than 100°C can be used if the
polypropylene slides or substrates deform in 100°C PFB. Temperatures below
100°C will work, albeit with lower efficiency than the 100°C buffer.
3. Incubate polypropylene slides
in 100°C PFB for 2 hours. This incubation step removes residual chemicals from
the polypropylene surface and functionalizes the surface to enhance binding of
antigens, antibodies and other proteins. Make sure to keep the plastic slides
submersed at all times during this step.
4. Rinse polypropylene slides
exhaustively with tap water for 5 minutes. This step should be performed using
a steady stream of tap water for at least 5 minutes to ensure that all PFB has
been removed from the polypropylene surface.
5. Heat Arrayit Polypropylene
Rinse Buffer (PRB) to 100°C. Heating can be performed in a microwave oven or on
a hot plate. Make sure to wear nitrile gloves and safety glasses at all times
when handling hot PRB.
6. Rinse polypropylene slides
once with PRB to remove residual tap water. This step removes residual tap
water from the surface prior to the final rinse step. Tap water contains ions
and other contaminants that can reduce the performance of immunoassays, and
such contaminants are removed by PRB during this step.
7. Incubate polypropylene slides
in 100°C PRB for 1 hour. Submerge the polypropylene slides in 100°C PRB and
incubate for one hour. The slides can be inverted periodically to make sure
that both sides are rinsed properly. Weight can be added to the slides to make
sure that the slides are fully submerged at all times. PRB can be used at
temperatures lower than 100°C, albeit with reduced efficiency compared to the
100°C buffer.
8. Dry polypropylene slides overnight
at 37°C. Remove the polypropylene slides from the PRB and dry them overnight
(12 hours) at 37°C in a drying oven. The drying step removes residual moisture
from the polypropylene surface, which increases protein binding efficiency.
Polypropylene Slide Protein Binding
Short Protocol
1. Remove the polypropylene slides
from the drying oven.
2. Print 1 µg/µl aqueous
protein samples onto the plastic slides.
3. Incubate overnight at
37°C to dry the proteins onto the plastic surface.
4. Place the polypropylene slides
in Arrayit BlockIt™ Blocking Buffer (BKT).
5. Block three times for 10
minutes in room temperature BKT with gentle mixing.
6. Total blocking time
should be 30 minutes (3 x 10 min).
7. After 30 minutes of
blocking, proceed immediately to the protein-protein binding step.
Polypropylene Slide Protein Binding
Complete Protocol
1. Remove the polypropylene slides
from drying oven. Place the slides on a microarray printer deck, lab bench or
other surface to be used for protein printing. All surfaces should be clean and
free of dust and other contaminants to avoid contaminating the slides. Wear
white nitrile gloves at all times when handling the functionalized
polypropylene slides.
2. Print 1 µg/µl of aqueous
protein samples onto the polypropylene surface. These proteins can be
antibodies, antigens, recombinant proteins, peptides, cell extracts and other
sources. Printing can be performed using a microarray printer, microarray
spotter, microarrayer or other suitable printing robot. Proteins can also be
deposited by hand using a micropipette. Polypropylene has a 1 µg/cm2
estimated protein binding capacity, so lower concentrations of protein (e.g.
0.1-1.0 µg/µl) may suffice to saturate the surface. Lower protein
concentrations may be useful in cases where the protein reagents are expensive,
and a dilution series should be performed to determine the precise optimal
protein concentration for a given assay.
3. Incubate overnight at
37°C to dry the protein droplets. This step removes water from the protein
droplets and concentrates the protein onto the polypropylene surface to enhance
binding. Printed slides should be handled carefully to avoid displacing the
droplets from printed surface. Drying times of less than 12 hours at 37°C may
suffice for protein binding, and should be optimized for a given immunoassay
based on throughput needs and other considerations. Protein binding to
polypropylene can be assessed using an appropriate protein staining reagent or
can be assessed as a decrease in contact angle in the printed area as shown in
Figure 1.
Figure 1.
Contact angle determination to assess protein binding to a 1.2 mm thick polypropylene
slide. The slide was functionalized using Arrayit Polypropylene
Functionalization Buffer (Cat. PFB), rinsed with Arrayit Polypropylene Rinse
Buffer (Cat. PRB) and printed using 1 µg/µl protein delivered with a 100 µl pipette.
Proteins were deposited onto a 0.5 cm2 area of the plastic slide
(blue arrows) and the slide was dried for 12 hrs at 37°C to allow protein
binding. A 20 µl droplet of distilled water was placed on the printed area or
on an adjacent slide locations. Protein binding to polypropylene reduces surface
hydrophobicity because proteins are hydrophilic, which causes a contact angle
decrease (less convex droplet) as shown above.
4. Place the polypropylene slides
in Arrayit BlockIt™ Blocking Buffer (BKT). Remove the polypropylene slides from
the 37°C drying oven and place them on a clean, dry lab bench. Submerge the
polypropylene slides in room temperature BKT, making sure the slides are
completely submerged. The blocking steps can be performed in Arrayit Microarray
Reaction Trays (Cat. MRT) or using other suitable hardware.
5. Block three times for 10
minutes in room temperature BKT with gentle mixing. Incubate the polypropylene slides
for 10 minutes in BKT and repeat two more times for a total of three blocking
steps. Blocking prevents non-specific protein binding during the protein-protein
binding steps (see below).
6. Total blocking time
should be 30 minutes (3 x 10 min). For high-throughput applications, lesser
blocking times 10-15 total minutes can be used to increase processing speed.
Lesser blocking times may result in higher non-specific binding, and should be
tested before finalizing the protocol for a given assay.
7. After 30 minutes of blocking,
proceed immediately to the protein-protein binding step. Proceeding quickly
ensures optimal activity of the protein (e.g. antibody, antibody) bound to the
polypropylene surface. Protein-protein binding can be assessed using a protein
staining reagent labeled with a fluorescent, colorimetric or other suitable
label, followed by scanning with a microarray scanner or imager. Macroscopic
assays may be scored visually depending on the assay design.
Polypropylene Slide Protein-Protein Binding
Short Protocol
1. Suspend labeled protein
in Arrayit Protein Microarray Reaction Buffer (PMRB).
2. Pipette the labeled
protein mixture onto the printed area.
3. Incubate for 30 minutes
at room temperature to allow protein-protein binding.
4. Wash five times for 1
minute with Arrayit Protein Microarray Wash Buffer (PMWB).
5. Rinse 1 time for 10
seconds with Arrayit Protein Microarray Rinse Buffer (PMNB).
6. Detect protein-protein
binding by scanning, imaging or visually.
Complete Protocol
1. Suspend labeled protein
in Arrayit Protein Microarray Reaction Buffer (PMRB). PMRB buffer contains
salts and detergents that facilitate protein-protein binding. The optimal
concentration of the labeled protein will depend on a large number of factors
including binding affinity and kinetics, background binding, and so forth. For
a fluorescent secondary antibody, 0.1-1 ng/µl is a good starting point for
optimization.
2. Pipette the labeled
protein mixture onto the printed area. Use a micropipette to transfer the
labeled antigen, antibody, extract or other labeled protein onto the printed protein
spots on the polypropylene surface. Care should be taken to avoid direct
contact between the pipette tip and the polypropylene surface as this can
damage the printed protein spots.
3. Incubate for 30 minutes
at room temperature to allow protein-protein binding. This step allows
protein-protein binding between the protein bound to the polypropylene surface
and the labeled proteins in solution. Elevating the temperature from room temperature
to 37°C, along with gentle mixing with agitation or vibration can be used to
enhance binding kinetics. Care should be taken not to displace the labeled
sample from the printed reaction area on the polypropylene surface.
4. Wash five times for 1
minute with Arrayit Protein Microarray Wash Buffer (PMWB). After the 30 minute
protein-protein binding step, the reaction area on the polypropylene surface should
be washed five times for 1 minute per wash to remove unbound labeled protein.
Use gentle agitation during the five washes.
5. Rinse 1 time for 10
seconds with Arrayit Protein Microarray Rinse Buffer (PMNB). After the five washes,
rinse the reaction area on the polypropylene slide once for 10 seconds using PMNB.
This step removes salts and detergents that can interfere with the detection
step.
6. Detect protein-protein
binding. Using fluorescent, colorimetric or label-free detection, scan or image
the reacted area to detect binding events between the proteins bound to the
polypropylene surface and those from the labeled solution specimen. The protein
assay should be optimized to maximize the signal-to-noise ratio (SNR), speed,
throughput and affordability of the assay.
Troubleshooting Tips
Protein binding efficiency
lower than expected
- Rinse polypropylene more
thoroughly after functionalization.
- Check printed protein
concentration.
- Increase drying time
and/or reduce relative humidity.
Low protein-protein binding
signals
- Optimize printed protein
concentration (lower or higher than current assay).
- Check activity or
secondary antibody or labeling reagent.
- Check scanner or imager
settings.
Recommended Products and Ordering Information
Arrayit Polypropylene
Functionalization Buffer, Cat. PFB, 1
Liter of 1X solution
Arrayit Polypropylene Rinse
Buffer, Cat. PRB, 1
Liter of 1X solution
Arrayit BlockIt™ Blocking
Buffer, Cat. BKT, 250
ml of 1X solution
Arrayit Protein Microarray
Reaction Buffer, Cat. PMRB, 250
ml of 1X solution
Arrayit Protein Microarray
Wash Buffer, Cat. PMWB, 1
Liter of 1X solution
Arrayit Protein Microarray
Rinse Buffer, Cat. PMNB, 500
ml of 1X solution
Arrayit Microarray Reaction
Trays, Cat. MRT, 10
per package
Arrayit SpotBot® 4 Pro
Personal Protein Edition Microarray Printer, Cat. SPA4PRO
Arrayit NanoPrint™ 2 LM60PRO
Enterprise Level Protein Microarray Printer, Cat. LM60PRO-2
Arrayit InnoScan® 710
Two-Color Fluorescence Microarray Scanner, Cat. 710
Warranty
Arrayit life sciences
products are sold for research purposes only. Arrayit brand products have been
scientifically developed and are sold for research purposes. Extreme care and
exact attention should be practiced in the use of the materials described
herein. All Arrayit brand products are subject to extensive quality control and
are guaranteed to perform as described when used properly. Any performance
issues should be reported to Arrayit immediately. Arrayit’s liability is
limited to the replacement of the product, or a full refund. Any misuse of this
product is the full responsibility of the user, and Arrayit makes no warranty
or guarantee under such circumstances. Pricing may vary up to 30% due to costs
associated with distribution, import taxes, duties, customs clearance and
shipping.