Arrayit offers the market’s most advanced and comprehensive human proteome microarray. HuProt™ v3.1 24K Human Proteome Microarrays contain  24,576 (24K) protein samples representing >20,000 human proteins and comprising 81% of the human proteome. HuProt™ v3.1 microarrays empower unprecedented human proteomics applications including global analysis of the human immune response, small molecule-protein interactions, enzyme-substrate interactions, antibody binding specificity, protein-protein interactions, autoantibody discovery and the binding of DNA and RNA to the human proteome. HuProt™ v3.1 glass substrate slide format ensures affordable deployment for research, pharmaceutical studies and diagnostic biomarker identification.

Product Features
Arrayit HuProt™ v3.1 24K Human Proteome Microarrays provide the world’s largest collection of human proteins in a high-performance and affordable glass microarray format. Users will appreciate the following product features:

• World’s most advanced and comprehensive human proteome microarrays
• Contains all major protein families including transcription factors, kinases and secreted proteins
• 24,576 (24K) protein samples printed onto glass slides
• >20,000 human proteins represent 81% of the human proteome
• 119 mouse proteins increases total proteins content to >20,000+
• Duplicate printing of all 24,576 proteins for 49,152 total printed spots
• Manufactured using advanced microarray printing technology
• Cold room manufacturing ensures the stability of HuProt™ v3.1 protein content
• Proprietary coupling chemistry and atomically polished glass substrate slides
• Universal 1 x 25 x 76 mm slide format compatible with >10,000 scanners worldwide
• Fluorescence detection enables both single and multi-color labeling
• Perform both absolute and relative protein expression quantification measurements
• Supported by Arrayit InnoScan® scanners and Mapix® software
• HuProt™ is supported by Arrayit’s entire microarray reagents and consumables product line
• Human proteins expressed in brewer’s yeast Saccharomyces cerevisiae
• Yeast recombinants ensure proper folding and post-translation modification
• GST fusion technology ensures high protein purity and easy quality control
• High protein density provides strong signals and low background
• Covalent protein coupling to the glass surface increases assay stability
• 100 µm diameter printed spots detectable using all standard microarray scanners
• Corner slide chamfer permits unambiguous slide orientation
• Barcode identification speeds sample tracking for large experiments
• Profile the human proteome for immune responsiveness
• Identify protein-small molecule interactions for drug discovery
• Determine the proteomic binding specificity of monoclonal and polyclonal antibodies
• Examine protein-protein interactions on a global scale
• Identify DNA and RNA binding proteins on a proteomic scale
• -80°C storage ensures extended 12-month HuProt™ stability
• Complete HuProt™ v3.1 24K Human Proteome Microarray Services available (Cat. HP24KS)

Figure 1. Arrayit HuProt™ v3.1 24K Human Proteome Microarrays contain 24,576 protein samples and >20,000 human proteins representing 16,152 human genes comprising all of the main protein functional families including transcription factors, kinases, metabolism, signal transduction, cell death, cell communication, secreted proteins, nuclear proteins and membrane proteins (left).  HuProt™ v3.1 24K Human Proteome Microarray expression clones were sequenced to verify protein coding sequence integrity and more than 89% correspond to full length human proteins (right).

Kit Contents

• HuProt™ 24K Human Proteome Microarray, 1 each
• HuProt™ 24 x 60 mm lifter slip coverslip, 1 each

Storage and Handling
HuProt™ microarrays must be stored in an ultra cold and dry environment. HuProt™ microarrays are shipped in closed plastic slide holders on dry ice or with gel coolant packs at -70°C. Upon arrival, microarrays should immediately be stored at -70°C. To ensure the best performance from the HuProt™ microarray:

• Wear nitrile gloves at all times.
• Do not touch the active surface of the microarray (the surface where the barcode label is attached) with hands, pipette tips or tweezers.
• The active surface should face up at all times during the assay.
• Handle microarrays on the edges and near the barcode end only.
• Do not allow HuProt™ microarrays to dry out at any time during the assay.
• For low volume assays, carefully place the glass lifter slip coverslip (minimizes evaporation) onto the active surface of the microarray.
• When the assay is complete, carefully remove the cover slip from the microarrays prior to the washing steps because surface scratches can damage the microarray. An  alternative is to immerse the microarray in a large volume of wash buffer, and allow the cover slip to float off naturally during the wash step.

Preparation for Use

• It is critical to keep the microarrays ultra cold and ultra dry right up until the time of use.
• HuProt™ microarrays should be stored at -80°C for best results. Before removing HuProt™ microarrays from storage, place a layer of dry ice pellets inside a styrofoam box with a lid and cover tightly. Remove the plastic slide holder containing HuProt™ microarrays from storage at -80°C and place it on top of the dry ice. Add an additional layer of dry ice pellets or a sheet of frozen gel coolant on top of the plastic slide holder. If dry ice is not available in your laboratory, transfer the HuProt™ microarrays quickly from -80°C and proceed immediately to the blocking steps.
• Prepare Microarray Reaction Trays (Cat. MRT) for the blocking steps. Microarray Reaction Trays hold the HuProt™ microarrays during the blocking, reaction (in the case of high volume samples) and washing steps. Each well of the Microarray Reaction Tray holds one HuProt™ microarray.
• Add 5.0 ml of blocking buffer to each compartment of the Microarray Reaction Tray. Handle the microarray on the edges of the glass slide and barcode end only. Be careful not to touch the active surface of the microarray.
• Block the HuProt™ microarrays. The active surface of the HuProt™ microarray is the surface with the barcode. Carefully use fine-nosed tweezers to remove one microarray from the plastic slide holder that is resting on dry ice. Immediately submerge the HuProt™microarray with the active surface facing up in a Microarray Reaction Tray containing 5.0 ml blocking buffer. Incubate with gentle shaking for 5 min. Carefully pour off the blocking buffer or remove it by aspiration. Add 5.0 ml fresh blocking buffer to the Microarray Reaction Tray and repeat the blocking steps three more times at 5 min each.
• Remove the blocking buffer from one corner of the Microarray Reaction Tray via aspiration and proceed immediately to the assay of choice. Blocking solutions vary by protocol, so please refer to the protocol of interest for the correct blocking solution.

Sample Preparation and Assays

• High volume assays. If a large sample volume is available, dilute it in 5.0 ml of reaction buffer. Add the 5.0 ml sample to one compartment of a Microarray Reaction Tray. Immerse the HuProt™
  microarray face up in the 5.0 ml of diluted sample. Coverslips are not required for 5.0 ml sample volumes.
• Low volume assays (100 μl). Evaporation must be minimized. Prepare 100 μl of sample in reactin buffer and add the sample to the active surface of the microarray. Immediately cover the microarray with a 24 x 60 mm lifter slip cover slip (Cat. CST2). Place the covered microarray in an Arrayit Hybridization Cassette (Cat. AHC) to ensure humidification during the reaction step.
• Washing. When binding reactions are complete, wash the microarrays in Microarray Reaction Trays using 4 wash cycles of 3 minutes per cycle with 4 ml wash buffer per well and gentle rocking or shaking. For low volume 100 µl reactions, remove the cover slip early in the first wash step using fine-nosed tweezers to prevent surface scratches. For high volume 5.0 ml samples, the washes are also performed in Microarray Reaction Trays using 4 wash cycles of 3 minutes per cycle with 4 ml wash buffer per well and gentle rocking or shaking.
• If fluorescent reagents are used in the assay, cover the reaction and wash trays with aluminum foil to prevent photobleaching.
• Scanning and storing reacted microarrays. After the wash steps are complete, dry the microarrays immediately by centrifugation for 3 sec in an Arrayit Microarray High-Speed Centrifuge (Cat. MHC110V or MHC220V). Scan the microarrays immediately (highly preferred) using an Arrayit InnoScan® 910, 1100, 710 or equivalent scanner. Microarrays may also be stored in slide boxes and sealed in anti-static bags at -20°C for up to three days, though diminished signals may be observed depending on the stability of the labeling reagents.

Antibodies and Quality Control

• Secondary antibodies for the detection step. The quality of commercially available secondary antibodies varies widely. Please test all secondary antibodies thoroughly before conducting your assay. The secondary antibodies listed in this manual are provided as examples.
• A fluorescent alignment grid for data analysis can be obtained using the GST epitopes contained within the recombinant proteins on HuProt™ microarrays. After the primary assay has been completed, an anti-glutathione-s-transferase antibody (e.g. EMD Millipore, Cat A-21428) can be used to detect HuProt™ microarray fusion proteins. GST antibodies should use a wavelength different from the primary assay to allow unambiguous detection. Please note that the GST signals obtained after your primary assay should not be used as a measure of the HuProt™ microarray quality because many of the proteins on the microarray will lose some activity during the primary assay. Make sure to dilute antibodies 1:1,000 to 1:10,000 to ensure strong signal strenght and low background. Excessive antibody concentrations will produce elevated background and should be avoided in all HuProt™ microarray assays.

Monoclonal Antibody Specificity Determination Assay
1. Store HuProt™ microarrays inside the closed plastic slide holders at -70°C or on a layer of dry ice right up until the blocking step. The active surface of the HuProt™ microarray is the surface with the barcode. It is critical to keep the HuProt™ microarrays ultra cold and ultra dry prior to use. Do not allow liquid to condense or fall onto the microarray surface as this will reduce microarray quality.

2. Add 5.0 ml of blocking solution to each compartment of an Arrayit Microarray Reaction Tray. Use fine-nosed tweezers to carefully remove one microarray from the plastic slide holder resting on dry ice. Immediately submerge the HuProt™ microarray with the active surface up in a compartment of the Microarray Reaction Tray containing 5.0 ml blocking buffer. Incubate with gentle shaking for 5 min. Carefully pour off the blocking buffer or remove by aspiration. Repeat this blocking step three more times for 5 min per step using 5.0 ml of blocking solution and gentle shaking at 37°C or room temperature.

3. Prepare the primary monoclonal antibody for testing. If the primary antibody is available in limited quantities, use a 100 µl reaction and a 24 x 60 mm lifter slip cover slip. Dilute the antibody to 400 ng/ml in 100 µl of Arrayit Microarray Reaction Buffer. If the primary antibody is available in large quantities, use a 5.0 ml reaction and an Arrayit Microarray Reaction Tray. Dilute the primary antibody to 400 ng/ml in 5.0 ml of Arrayit Microarray Blocking Buffer. If the antibody is from tissue culture medium, prepare 100 µl of a 1:12 dilution in Arrayit Microarray Reaction Buffer. The 1:12 dilution assumes that the antibody concentration is 0.005 mg/ml in the supernatant. Supernatant dilutions should be adjusted accordingly when using more or less concentrated supernatants.

4. Add the monoclonal antibody sample to the HuProt™ microarray. For 5.0 ml reactions, use aspiration to carefully remove the blocking buffer from a corner of the Microarray Reaction Tray. Avoid touching the microarray surface at all times during the reaction step. For low volume 100 μl samples, pipette 100 µl of the 400 ng/µl sample directly onto the active surface of the blocked HuProt™ microarray (barcode facing upward) and cover the microarray with a 24 x 60 mm lifter slip cover slip. Place the microarray in an Arrayit Microarray Hybridization Cassette and react for 60 min at 37°C or room temperature. For high volume samples, add 5.0 ml of sample to a Microarray Reaction Tray well, place the HuProt™ microarray in the well, and mix for 60 min at 37°C or room temperature with gentle shaking.

5. Following the reation step, wash the microarrays with Arrayit Protein Microarray Wash Buffer. For low volume 100 µl reactions, the cover slip must be removed early in the first wash step. Place the microarray in a well of the Microarray Reaction Tray and add 5.0 ml of Protein Microarray Wash Buffer. Use fine-nosed tweezers to carefully remove the lifter slip cover slip. An alternative method is to float the cover slip off by immersing the microarray in a larger volume of Protein Microarray Wash Buffer. Wash in 5.0 ml of Protein Microarray Wash Buffer for 2 min at 37°C or room temperature. Carefully remove the wash buffer by pouring or aspiration. Repeat the wash steps six more times.

6. After the wash steps, prepare the secondary antibody solution. Dilute the secondary detection antibody in Arrayit Microarray Reaction Buffer at a 1:1,000 to 1:10,000 dilution. Add 5.0 ml of diluted secondary antibody solution to each compartment of an Arrayit Microarray Reaction Tray. Cover the reaction tray with aluminum foil and incubate at 37°C or room temperature for 30 min with gentle shaking.

7. Following the secondary antibody reaction step, remove the buffer from a corner of the reaction tray by pouring or aspiration. For fluorescent assays, microarrays should be protected from light at all times to minimize photobleaching. Add 5.0 ml of Arrayit Microarray Wash Buffer per reaction tray well and wash for 2 min at 37°C or room temperature with gentle shaking. Remove the wash buffer by pouring or aspiration and repeat this wash step six more times using 5.0 ml wash buffer per wash for 2 min at 37°C or room temperature with gentle shaking.

8. Following the secondary antibody wash steps, add 5.0 ml of Arrayit Microarray Rinse Buffer and rinse for 1 min at 37°C or room temperature with gentle shaking. Repeat this rinse step one more time with 5.0 ml of rinse buffer for 1 min at 37°C or room temperature with gentle shaking.

9. Following the rinse steps, blot the edge of the HuProt™ microarray on an Arrayit Microarray Cleanroom Wipe to remove excess rinse buffer, immediately place the microarray in an Arrayit Microarray High-Speed Centrifuge and dry by centrifugation for 3 sec.

10. Following the dry step, place the microarray immediately into the Arrayit InnoScan® Microarray Scanner and scan using the laser and PMT settings that saturate fewer than 1% of the printed 19K elements. Microarrays can also be stored in sealed holders or slide boxes at -20°C or -80°C for up to three days before scanning, though diminished signal intensities may be observed. Save a 16-bit TIFF image from one or more scan settings and quantify the data using Arrayit InnoScan® Mapix® Quantification Software. The quantified  HuProt™ microarray data can be used in downstream software for data analysis and mining.

Additional Materials and Equipment

• Arrayit InnoScan® 910, 1100 and 710 Microarray Fluorescence Scanners
• Arrayit High-Speed Microarray Centrifuge, Cat. MHC110V and MHC220V
• Arrayit Microarray Hybridization Cassettes, Cat. AHC
• Arrayit Microarray Reaction Trays, Cat. MRT
• Arrayit Microarray Substrate Slide Tubes, Cat. MST
• Arrayit 24 x 60 mm LifterSlip™ Cover Slips, Cat. CST2
• Arrayit Protein Microarray Buffer Kit, Cat. PMBK
• Arrayit Microarray Cleanroom Wipes, Cat. MCW
• Arrayit Microarray Cleanroom Gloves, Cat. MCGL
• Aluminum foil
• Automatic pipettes
• Micropipettes
• Orbital shaker
• Sterile disposable micropipette tips
• Sterile serological pipettes
• Fine-nosed tweezers
• Vacuum aspiration system
• Vortex