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Protoarray™ Protein Microarrays—General

The main reasons for using protein array technology are:

  • Speed
  • Sensitivity
  • Throughput
  • Cost
  • Open platform technology

A single microarray slide can accommodate thousands of individual proteins that can be screened in a single experiment against a variety of molecules. Since assay time is short and only a very small sample amount is required, a wealth of information can be generated extremely quickly without a significant investment. In addition, protein array technology can address protein interactions with a variety of molecules (proteins, lipids, sugars, DNA, RNA, antibodies, small molecules, etc.), which is something that no other single technology platform can accomplish. Protein arrays are therefore excellent lead screening tools for basic science research and drug discovery and development.

The Protoarray™ Microarray allows you to: 

  • Detect novel protein-protein interactions 
  • Validate previously observed protein-protein interactions for PPI (protein protein-interaction) applications or observed signals for KSI (kinase substrate identification) applications 
  • Confirm positive interactions using the identified interacting protein on the array as a probe in reciprocal experiments 
  • Test various experimental conditions for the protein interactions or your kinase 
  • Rapidly perform serum profiling using a sensitive method to discover potential autoantigen biomarkers 
  • Identify potentially biologically relevant protein kinase substrates, small molecule binding partners, ubiquitin ligase substrates, and protein interactors of research or therapeutic antibodies 

The Protoarray™ Human Protein Microarray v5.1 specifications are listed below:

  • Total Subarrays: 48 (4 columns × 12 rows) 
  • Subarray Size: 4,400 μM × 4,400 μM 
  • Subarray Dimensions: 22 rows × 22 columns 
  • Median Spot Diameter: ~110 μM 
  • Spot Center to Center Spacing: 200 μM 
  • Distance Between Subarrays: 100 μM 
  • Replicates per Sample: 2 
  • Total Human Proteins on v5.1 Array: >9000

The proteins on the microarray are printed in 48 subarrays that are equally spaced in vertical and horizontal directions. Each protein is printed in duplicate. For details on the human protein and control spots on the Protoarray™ Human Protein Microarray v5.1, go here.

The slides are 1 inch x 3 inch (25 mm x 75 mm) in dimension.

The barcode on the Protoarray™ Protein Microarray slide is lot-specific and can be used to track samples and to retrieve lot-specific array information by going here.

The Protoarray™ Human Protein Microarray v5.1 is shipped on dry ice.

We recommend storing the Protoarray™ Protein Microarrays at -20 degrees C. Some additional guidelines are as follows:

  • Do not store arrays in extremely humid conditions.
  • Always keep slides in sealed containers prior to use.
  • Do not add desiccant during storage.

Note: An expiration date is printed on the packaging for the microarray. We recommend using the array before expiration for the best results.

To ensure reproducibility and an acceptable signal-to-noise ratio, we recommend a single use for each Protoarray™ Protein Microarray. We do not recommend re-using the array or re-probing the same array with another probe.

To obtain the best results, all incubations of the Protoarray™ Microarray with various solutions are performed in a 4-chamber, covered incubation tray that can be purchased from Sarstedt (Cat. No. 94.6077.307).

Note: Incubation trays or other hybridization chambers are not suitable for use in the probing portion of the KSI or SMI-radioactive application, as you need a container that seals tightly to prevent any leakage of radioactive material during the washing steps. 

We recommend using coverslips that are able to completely cover the printed area (20 mm × 60 mm) of the glass slide and hold a small reagent volume to minimize the amount of valuable probe used and prevent evaporation of reagents. We recommend using glass coverslips (VWR, Cat. No. 48404-454). 

Yes, we offer 10X Synthetic Block, Cat. No. PA017.

For a complete list of our Protoarray™ custom service offerings, description of the service and inquiry form, go here.

Protoarray™ Protein Microarrays—Protein Content

The Protoarray™ Human Protein Microarray v5.0 and Control Protein Microarray v5.0 have been discontinued. The alternative is the Protoarray™ Human Protein Microarray v5.1.

The Protoarray™ Human Protein Microarray v5.1 contains >9000 human proteins with each protein being printed in duplicate. The full list of proteins can be found here. It also contains various control proteins, the full list of which can be found on Pages 7-8 of the manual

The Protoarray™ Human Protein Microarray v5.0 contained 9318 human proteins with each protein being printed in duplicate. The full list of proteins can be found here. The Protoarray™ Control Protein Microarray v5.0 contained 2016 control proteins with each protein being printed in duplicate. The Protoarray™ Human Protein Microarray v5.0 and Control Protein Microarray v5.0 have been discontinued. The alternative is the Protoarray™ Human Protein Microarray v5.1, which also includes >9,000 human proteins. The full list of proteins for version 5.1 can be found here

Below is a table showing the different classes of proteins spotted on the Protoarray™ Human Protein Microarray v5.1. 

Class

Number of proteins

Protein kinases

729

Transcription factors

337

Membrane proteins

1651

Nuclear proteins

1072

Signal transduction

1966

Secreted proteins

199

Cell communication

997

Metabolism

4324

Cell death

936

Cell surface

194


Note: The Protoarray™ Human Protein Microarray v5.0 and Control Protein Microarray v5.0 have been discontinued. The alternative is the Protoarray™ Human Protein Microarray v5.1.

The majority of the human protein collection on the Protoarray™ Human Protein Microarray v5.1 is derived from our Human Ultimate ORF Clone Collection. The ORFs are expressed in Sf9 insect cells as N-terminal GST fusion proteins using a Gateway-adapted baculovirus expression vector. 

Each Ultimate™ ORF Clone is full insert sequenced and is guaranteed to match the corresponding GenBank™ amino acid sequence. Some of the human proteins printed on the array represent the human protein kinase collection derived from full insert sequenced clones but are not Ultimate™ ORF Clones. Some of the kinases from the kinase collection have been cloned as catalytic domains rather than full-length proteins. About 310 proteins printed on the array are derived from our purified protein kinase collection. Approximately 40 additional proteins printed on the array are purified cytokines available to us. Approximately 20 proteins, peptides, and nucleic acids that have been demonstrated to be antigens in a variety of autoimmune diseases are also printed on the array. Content for Protoarray™ v.5.1 arrays was enriched for proteins relevant to disease processes, for a total of >6,100 potential drug targets printed on the array. 

The expressed proteins are purified by affinity chromatography under high-throughput conditions optimized to obtain maximal protein integrity, function, and activity. Following purification, each protein is assayed for purity and expected molecular weight. Protein arrays are manufactured at 4 degrees C and stored immediately at -20 degrees C.

For accession number and amino acid sequence for each protein as well as information on peptides and nucleic acids printed on the array, download the Protein Content List here.

The amount of each protein on the ProtoArray™ microarray is determined by probing representative arrays from each lot with an anti-GST antibody. The signal observed for each protein is then normalized and reported as relative RFU. This measure is not an absolute amount of protein but reflects the relative intensity of the signal seen for each of the GST-tagged proteins on the array. 

The volume of protein solution for each spot is approximately 0.5 to 1.0 nL. Not all proteins have the same affinity for the ultrathin nitrocellulose coating of the slides. Approximately 10% or less of the protein binds to the surface of the array. Please note that this is intended as anecdotal information only, you should not use this information to draw any firm conclusions about the amount of protein present in your data analysis.

Yes, membrane proteins are well-represented on the Protoarray™ Human Protein Microarray v5.1. 

The proteins on the Protoarray™ Human Protein Microarray should be stable for at least 6 months if the microarrays are stored as recommended.

Note: An expiration date is printed on the packaging for the microarray. We recommend using the array before expiration for the best results.

After purification, a sample of every protein is assayed. Proteins are an average of >90% pure as determined by Coomasie™ blue stain or capillary electrophoresis. 

Protease inhibitors are used during the protein purification steps. All the proteins are expressed and purified at 4 degrees C. The protein arrays are manufactured at 4 degrees C and stored immediately at -20 degrees C. Phosphospecific antibody profiling experiments have shown that the proteins retain their posttranslational modifications.

Only the Alexa Fluor™ dye–labeled control proteins have autofluorescence. These proteins are used as positive controls for fluorescence scanning and for orientiation of the microarray image. None of the other proteins on the Protoarray™ Protein Microarray have any autofluorescence.

The Control Kinase (Cat. No. PV3304) is a recombinant human MAPK14 p38 alpha purified from E. coli. It serves as a control to probe the Protoarray™ Human Protein Microarray v5.1 for KSI experiments to help to determine which signals are specific to your kinase. 

Note: You can also probe the array using your kinase of interest.

Protoarray™ Protein Microarrays—Protein Labeling, Probing and Detection

The recommended protein probe concentration range for probing the Protoarray™ Human Protein Microarray is 100 nM–10 μM for biotinylated proteins and 10 nM–1 μM for V5-tagged proteins. A number of options are available for probing the Protoarray™ Human Protein Microarray v5.1 with the protein probe of interest using pre-made reagents, or your own buffers and detection reagents. Probing options can be performed individually, or in tandem, and include: 

  • Probing with your protein probe to detect novel interactions. 
  • Probing with only the detection reagent (negative control). The negative control allows you to determine signals specific to your probe. 
  • Probing with different probe concentrations to determine the optimal amount of probe for your assay. Start with an initial probe concentration. If the initial signal is strong with low background, confirm the initial results with a second array using the same experimental conditions. If the initial results indicate weak signal or an unacceptable signal-to-noise ratio, probe a second array with a different probe concentration as described below: 
 Probe first array….  And….  Then probe second array…. 

With 10 nM probe 

Weak signal

With 1–10 μM probe

With 10 μM probe High background With 10–100 nM probe

To probe the Protoarray™ Protein Microarray, you need to have a purified protein with a suitable tag. You can use proteins purified under native conditions, from E. coli, yeast cells, or higher eukaryotes. The protein of interest can be tagged using an epitope tag or a biotin label. 

Epitope tag

Using an epitope tag at the N- or C-terminus of the protein probe allows the use of the recombinant fusion protein directly as a probe without any further modification wherein the tag is used as the marker for detection of interactions. The recommended epitope tag is the V5-epitope tag at the N-or C-terminus of the protein to obtain the best results. Epitope tags such as FLAG, myc, or HA can also be used for probing the microarray in conjunction with an appropriately labeled antibody.

Note: Do not use an anti-GST antibody or anti-polyhistidine antibody for detecting interactions on a Protoarray™ Protein Microarray, as the majority of proteins on the array are GST tagged, with some that are also polyhistidine tagged.

To generate your protein probe with an epitope tag, you need to express your protein of interest as a fusion protein in an expression vector containing the desired epitope tag at the N- or C-terminus of the protein.

Biotin Tag

The extremely high affinity of the biotin-streptavidin interaction makes biotin-protein conjugation an attractive method for probe labeling. Small amounts of the protein can be efficiently in vitro biotinylated in a simple procedure. The biotinylated protein probe is detected using a streptavidin detection system.

The recommended protein probe concentration range for probing the Protoarray™ Human Protein Microarray is 100 nM –10 μM for biotinylated proteins, and 10 nM – 1 μM for V5-tagged proteins. 

We recommend purifying the protein of interest under native conditions. If the protein is to be labeled by in vitro biotinylation then it should be > 90% pure as impurities that also undergo biotinylation may result in high background during probing of the array. Based on our experience, protein purification using glutathione (for GST) is better than using Ni-column (for 6xHIS). Generally, GST tagged proteins tend to be much more soluble and are easier to purify to >90% purity using non-denaturing purification.

We offer a variety of Alexa Fluor™ 647 conjugated secondary antibodies that you can use for fluorescent detection, in conjunction with your anti-FLAG primary antibody.

We recommend purifying the protein under native conditions and resuspending in a buffer that does not contain any primary amines such as ammonium ions, Tris, glutathione, imidazole, or glycine. If the buffer contains primary amines, sufficiently dialyze the protein against 50 mM HEPES buffer, pH 7.4 containing 100 mM NaCl, or PBS. For proteins purified using metal chelating column chromatography (ProBond™ resin or Ni-NTA resin), perform dialysis against two changes of PBS to significantly lower the imidazole concentration. The presence of high salt (500 mM NaCl), glycerol or non-ionic detergent should not affect the biotinylation reaction.

The probing buffer is a PBS-based buffer. Other probing buffers such as HEPES-based buffers can be substituted for PBS. We think it is unlikely that this will negatively affect the probing. We strongly recommend that buffers other than those recommended first be tested for use on the Protoarray™ Human Protein Microarray slide.

Yes. The majority of our validation experiments were performed with biotinylated GST-fusions. A GST-tagged protein is fine, but GST detection is not recommended, because nearly all the proteins on Protoarray™ Protein Microarrays have GST-tags.

The minimum recommended size of the protein probe when using the in vitro biotinylation kit is 15 kDa. The main reason for the size limit is to ensure that the biotinylated probe gets separated efficiently from the unincorporated biotin (which remains in the column) as the mixture passes through the resin.

We offer the Biotin-XX Microscale Protein Labeling Kit (Cat. No. B30010), which provides a convenient means for biotinylating small amounts (20-100 μg) of purified protein. This kit has been optimized for labeling proteins with molecular weights between 12 and 150 kDa, and contains everything needed to perform three labeling reactions and to separate the resulting conjugates from excess reactive biotin. We also offer the FluoReporter™ Biotin Quantitation Assay Kit for biotinylated proteins (Cat. No. F30751), which provides a sensitive fluorometric assay for accurately determining the number of biotin labels on the protein. The assay is based on the displacement of a ligand tagged with a quencher dye from the biotin binding sites of Biotective™ Green reagent.

In theory, a biotinylated peptide can be used to probe the array; however we have not developed or validated protocols for this type of probe and hence do not have any specific recommendations. A big consideration when using small peptides (~20 amino acids) as probes is the possibility of high background due to binding of the peptides to the nitrocellulose. A potential way to address this issue is by adding a nonrelated peptide sequence as a non-specific competitor, which could function to block sites that are not blocked by the blocking agent in the blocking buffer.

The recommended protein kinase concentration for probing the Protoarray™ Human Protein Microarray is 50 nM. 

A number of options are available for probing the human microarray with the protein kinase of interest using pre-made reagents, or your own buffers and detection reagents as described below. Probing options can be performed individually, or in tandem, and include: 

  • Probing with your kinase of interest at 50 nM with [γ-33P]ATP to identify potential substrates
  • Probing with only the buffer and no kinase (negative control) in the presence of radiolabeled [γ-33P]ATP. The negative control allows you to determine signals specific to your probe. 
  • Probing with MAPK14p38 alpha (positive control). The result from the positive control helps to determine which signals are specific to your kinase. 
  • Probing with different probe concentrations to determine the optimal amount of probe for your assay. Start with an initial probe concentration. If the initial signal is strong with low background, confirm the initial results with a second array using the same experimental conditions. If the initial results indicate weak signal or an unacceptable signal-to-noise ratio, probe a second array with a different probe concentration as described below: 

     Probe first array….  And….  Then probe second array…. 

    With 10 nM probe 

    Weak signal

    With 50–100 nM probe

    With 50 nM probe  High background With 1–10 nM probe

  • Note: Do not use [γ32P]ATP for the assay, use [γ33P]ATP as the use of [γ33P]ATP supports increased signal resolution during data acquisition. While [γ32P] ATP can be used for the assay, data quantitation with [γ32P]ATP is not supported. Do not use cold ATP for the kinase probing steps. If your kinase is stored in a buffer containing ATP, make sure the final concentration of cold ATP is less than 100 nM during the kinase probing step.

The recommended small molecule probe concentration for probing the Protoarray™ Human Protein Microarray is at least 2.5 μM. 

A number of options are available for probing the Protoarray™ Human Protein Microarray with your own buffers and detection reagents as described below. Probing options can be performed individually, or in tandem, and include: 

  • Probing with your small molecule probe to detect novel interactions
  • Probing with only the detection reagent (negative control). The negative control allows you to determine signals specific to your probe
  • Probing with different probe concentrations to determine the optimal amount of probe for your assay. Start with an initial probe concentration. If the initial signal is strong with low background, confirm the initial results with a second array using the same experimental conditions. If the initial results indicate weak signal or an unacceptable signal-to-noise ratio, probe a second array with a different probe concentration. 

The small molecule of interest can be tagged using a reactive Alexa Fluor™ dye or a biotin label. 

Alexa Fluor™ tag: Using amine- or sulhydryl-reactive Alexa Fluor™ dyes, small molecules with the appropriate functional group can be directly labeled for use as a probe. We recommend the use of reactive Alexa Fluor™ 647 to obtain the best results. To label your small molecule probe with an Alexa Fluor™ tag, your small molecule of interest must contain the appropriate functional group which will allow labeling with a reactive Alexa Fluor™ dye. 

Note:

  • When performing fluorescence detection, it is important to avoid exposing the array to light after probing with a fluorescent detection reagent. 
  • If performing direct labeling, always verify that labeling does not affect the binding affinity of the antibody. 
  • Although Alexa Fluor™ 555 or Cyanine 3 dyes can be used for detection, using them may result in higher background signals. 

Biotin tag: The extremely high affinity of the biotin-streptavidin interaction makes biotin-protein conjugation an attractive method for probe labeling. You may use any method to biotinylate your small molecule of interest. To label your small molecule probe with a biotin tag, your small molecule of interest must contain the appropriate functional group for labeling. The biotinylated small molecule probe is detected using a streptavidin detection system. 

The recommended small molecule probe activity range for probing the Protoarray™ Human Protein Microarray is 50 pCi/μL - 50 nCi/μL, with weaker interactions requiring activity of 10–50 nCi/μL. 

A number of options are available for probing the human microarray with a small molecule of interest using your own buffers and detection reagents as described below. Probing options can be performed individually, or in tandem, and include: 

  • Probing with your tritiated small molecule of interest to identify potential substrates.
  • Probing with 3H estradiol (positional mapping reagent). The result from the positional mapping reagent can serve as a positive control to help determine signals specific to your probe. 
  • Probing with different probe concentrations to determine the optimal amount of probe for your assay. Start with an initial probe concentration. If the initial signal is strong with low background, confirm the initial results with a second array using the same experimental conditions. If the initial results indicate weak signal or an unacceptable signal-to-noise ratio, probe a second array with a different probe concentration. 

A number of options are available for probing the Protoarray™ Human Protein Microarray with your own buffers and detection reagents as described below. Probing options can be performed individually, or in tandem, and include: 

  • Probing with your ubiquitination enzymes to detect novel interactions. 
  • Probing with only the detection reagent (negative control). The negative control allows you to determine signals specific to your probe. 
  • Probing with different probe concentrations to determine the optimal amount of probe for your assay. Start with an initial probe concentration. If the initial signal is strong with low background, confirm the initial results with a second array using the same experimental conditions. If the initial results indicate weak signal or an unacceptable signal-to-noise ratio, probe a second array with a different probe concentration. 

While it is possible to generate your own biotin-tagged probe, we recommend using LanthaScreen™ Biotin-Ubiquitin (Cat. No. PV4379 or PV4380). Because the lysine residues are unmodified during the labeling process, these labeled ubiquitin reagents are readily incorporated into ubiquitin-protein conjugates and poly-ubiquitin chains.

While it is possible to generate your own biotin-tagged probe, we recommend using LanthaScreen™ Biotin-Ubiquitin (Cat. No. PV4379 or PV4380). Because the lysine residues are unmodified during the labeling process, these labeled ubiquitin reagents are readily incorporated into ubiquitin-protein conjugates and poly-ubiquitin chains. 

A number of options are available for probing the Protoarray™ Human Protein Microarray with your own buffers and detection reagents as described below. Probing options can be performed individually, or in tandem, and include: 

  • Probing with your serum or plasma probe to detect novel interactions. 
  • Probing with only the detection reagent (negative control). The negative control allows you to determine signals specific to your probe. 
  • Probing with different serum or plasma concentrations to determine the optimal amount of sample for your assay. Start with an initial sample concentration. If the initial signal is strong with low background, confirm the initial results with a second array using the same experimental conditions. If the initial results indicate weak signal or an unacceptable signal-to-noise ratio, probe a second array with a different serum or plasma concentration. 

A number of options are available for probing the Protoarray™ Human Protein Microarray with your own buffers and detection reagents as described below. Probing options can be performed individually, or in tandem, and include: 

  • Probing with your antibody probe to detect novel interactions. 
  • Probing with only the detection reagent (negative control). The negative control allows you to determine signals specific to your probe. 
  • Probing with different antibody concentrations to determine the optimal amount of antibody for your assay. Start with an initial antibody concentration. If the initial signal is strong with low background, confirm the initial results with a second array using the same experimental conditions. If the initial results indicate weak signal or an unacceptable signal-to-noise ratio, probe a second array with a different antibody concentration. 

Note: 

  • The recommended primary antibody concentration range for probing each array is 0.1–10 μg/mL. Dilute concentrated antibody in Washing Buffer. 
  • Secondary Alexa Fluor™ 647 conjugates should be diluted to 1 μg/mL in Washing Buffer. 

Fluorescence detection is used to detect protein-protein interactions on Protoarray™ Protein Microarrays. Fluorescent detection offers high sensitivity, low background, and signal stability.

  • To detect an epitope tag on your protein probe, use a labeled antibody specific to the tag. The antibody can be directly labeled with a fluorescent dye or detected through a secondary antibody conjugated to a fluorescent dye.
  • To detect a biotin label on your protein probe, use streptavidin conjugated to a fluorescent dye for signal amplification and increased sensitivity.

The Alexa Fluor™ detection system is our recommended fluorescent detection method. The Alexa Fluor™ 647 fluorophore is brighter and more stable than other commercially available dyes, such as Cyanine 5 dyes, and is more sensitive for detecting interactions on protein arrays. We have demonstrated that detection with Alexa Fluor™ 647 produces approximately 2-fold higher signal/background ratios than Cyanine 5 detection. The excitation for Alexa Fluor™ 647 dye is 650 nm and emission is 668 nm. The excitation of 635 nm mentioned in the manual (Page 13) was chosen for use with the GenePix™ 4000B scanner that is used in-house. 

  • When performing fluorescence detection, it is important to avoid exposing the array to light after probing with a fluorescent detection reagent.
  • If performing direct labeling, always verify that labeling does not affect the binding affinity of the probe.
  •  Although Alexa Fluor™ 555 or Cyanine 3 dyes can be used for detection, using them may result in higher background signals.

Radioactive detection (33P or 3H) is also very sensitive and may be used for the Kinase Substrate Identification (KSI) or Small Molecule Interaction (SMI) profiling applications. For radioactive detection, both phosphor imaging and autoradiography will work well. The phosphor imager or autoradiography film must have resolution of at least 50 micron. The silver grain on most X-ray film is smaller than 50 micron and most common desktop scanners allow for at least 50 micron resolution scanning. For tritiated small molecule profiling, a tritium-sensitive phosphor screen should be used. The typical exposure time for kinase substrate profiling is 16–18 hours. For titrated small molecule and methlytransferase substrate profiling assays the typical exposure time is two weeks. A minimum resolution of 600 dpi should be used when acquiring data with a phosphor imager.

Other methods of detection include colorimetric and chemiluminescent detection, but they are not as sensitive. For chemiluminescent detection, streptavidin–HRP or anti-biotin-HRP can be used to detect the biotinylated probe. Chemiluminescence cannot be detected with a phosphor imager; the image must be acquired on X-ray film or CCD-based detection. 

Protoarray™ Protein Microarrays—Scanning and Data Analysis

The .GAL (GenePix Array List) file describes the location and identity of all spots on the protein microarray and is used with the microarray data acquisition software to generate files that contain pixel intensity information for all features on the array. You can download the .GAL file here by entering the complete Barcode number listed on the slide. Ignore leading zeros and enter the number starting with the first non-zero digit.

Note: The .GAL files are text files that contain the data in a format specified by GenePix™ Pro Microarray data acquisition software. If you are using any other microarray data acquisition software, you can use data from the .GAL files to generate files that are compatible with your microarray data acquisition software.

You can download the lot specific information for your Protoarray™ Microarray by going here. Enter the complete Barcode number listed on the slide to retrieve the information. Ignore the leading zeros and enter the number starting with the first non-zero digit.

The lot specific information includes:

  • GAL File—defines spot locations and identities of all proteins on the array and is essential for data acquisition by microarray image analysis software. If you are using software other than Molecular Device's GenePix™ Pro, check the software manual for file-type compatibility. This file also includes the detected protein concentration information in relative fluorescent units for each spot.
  • Protein Information File—provides a listing and description of the human proteins on the array. 
  • Protein Sequence File —this file includes Accession Numbers, FASTA headers and amino acid sequences of human proteins on the array. 
  • Controls Information File—provides information on the array controls including name, molecular weight, and concentration.

Data acquisition software is used to obtain pixel intensity information for each spot/feature on the array. Microarray data acquisition software such as GenePix™ Pro v6 or higher (Molecular Devices Corporation) or ScanArray™ Acquisition Software (PerkinElmer, Inc.) is suitable for data acquisition. To acquire Protoarray™ data from images generated from radiolabeled assays (KSI and SMI), you may also use Protoarray™ Prospector Imager 5.2 which is included with Protoarray™ Prospector v5.2 Analyzer Software. It is available for download. ProtoArray™ Prospector Imager is not designed for image analysis of fluorescence based applications. ProtoArray™ Prospector Imager does not contain functions specific for fluorescent scanner image analysis, such as spot shape detection. Microarray analysis software that is supplied with the fluorescent scanner, such as GenePix™ Pro should be utilized for image analysis of fluorescence based assays. 

The Protoarray™ Prospector v5.2 analyzer software quickly analyzes the data acquired from the Protoarray™ Prospector Imager or other image acquisition software and easily identifies statistically significant hits, saving time and effort. The Protoarray™ Prospector v5.2 Analyzer software is designed to analyze data and identify potential protein binding partners with a low false positive rate as compared to performing manual calculations using a spreadsheet program. In addition, the Protoarray™ Prospector analyzer software has features that allow you to modify the analysis method and compare data obtained from different microarrays. The Protoarray™ Prospector software is available free-of-charge to Protoarray™ Microarray users. It can also be downloaded here

The Protoarray™ Prospector software also accepts the output files (.GPR) generated by the GenePix™ Pro microarray data acquisition software and analyzes the data using specified algorithms to generate a list of human proteins showing significant interactions with the probe. If .GPR files are not available, consult the Protoarray™ Prospector User Manual for guidelines to format a results file that is compatible for import into Protoarray™ Prospector.