Frequently Asked Questions

The HuProt microarray and subsets of the HuProt content (Focus/custom microarrays and individual proteins) have been used successfully in a wide range of applications, which include:

Serum Profiling

• Le Voyer, T., Parent, A.V., Liu, X. et al., Autoantibodies against type I IFNs in humans with alternative NF-αB pathway deficiency. Nature.
• Malle, L., Patel, R.S., Martin-Fernandez, M. et al., Autoimmunity in Down’s syndrome via cytokines, CD4 T cells and CD11c+ B cells. Nature.
• Chaojun Hu, Yongzhe Li. et al., Autoantibody Profiling on Human Proteome Microarray for Biomarker Discovery in Cerebrospinal Fluid and Sera of Neuropsychiatric Lupus. PLOS One.<

Antibody Cross-reactivity Testing

• Anand Venkataraman, Seth Blackshaw. et al., A toolbox of immunoprecipitation-grade monoclonal antibodies to human transcription factors. Nature.

Protein-Protein Interaction

• Hyun Mi Kang, Jung Hwa Lim, et al., FBXL17/spastin axis as a novel therapeutic target of hereditary spastic paraplegia. Cell & Bioscience.
• Rui-Ping Deng, Sheng-Ce Tao, et al., Global identification of O-GlcNAc transferase (OGT) interactors by a human proteome microarray and the construction of an OGT interactome. Proteomics.

Protein-DNA Interaction

• Shaohui Hu, Heng Zhu, et al., Profiling the Human Protein-DNA Interactome Reveals ERK2 as a Transcriptional Repressor of Interferon Signaling. Cell.

Protein-RNA Interaction

• Baochang Fan, Chien-Sheng Chen, et al., A Human Proteome Microarray Identifies that the Heterogeneous Nuclear Ribonucleoprotein K (hnRNP K) Recognizes the 5′ Terminal Sequence of the Hepatitis C Virus RNA. Molecular & Cellular Proteomics.

Post-translational Modification

• Eric Cox, Heng Zhu, et al., Identification of SUMO E3 Ligase-Specific Substrates Using the HuProt Human Proteome Microarray. Proteomic Profiling.

Protein-Small Molecule Interaction

• Hai-nan Zhang, Sheng-ce Tao, et al., Systematic identification of arsenic-binding proteins reveals that hexokinase-2 is inhibited by arsenic. PNAS.
• Daohui Wang, Yuan Luo, et al., Meldonium Ameliorates Hypoxia-Induced Lung Injury and Oxidative Stress by Regulating Platelet-Type Phosphofructokinase-Mediated Glycolysis. Frontiers in Pharmacology.

If the HuProt microarrays are properly stored at -80˚C, the arrays have a shelf life of at least 6 months for most applications. For serum profiling, the arrays are good for at least one year after the date of manufacture. The arrays should be stored at -80˚C, and they are shipped on dry ice.

The proteins printed on the arrays are very stable if the arrays are stored at -80˚C. We have performed studies using arrays that are 12 months old and still observed significant binding signals.

User manual and a MSDS are available for HuProt microarrays here: https://cdilabs.com/content/literatures/user-manual-huprot-microarray.

Yes, HuProt microarrays are used primarily for in vitro research and are CGMP compliant.

Please contact us – we will be happy to discuss your inventory needs with you.

HuProt microarrays are placed in plastic slide mailers, which are in turn securely boxed and shipped on dry ice.

If you need a copy of the commercial invoice for customs, please let us know and we will send you a copy in advance.

For the full list of proteins by name and category please visit the resources section here: https://cdilabs.com/content/literatures/huprot-v40-content.

Standard HuProt microarrays are printed on nitrocellulose coated PATH™ slides. Other surfaces may be available, depending on your needs. The proteins are not bound/attached to the slide by any tags. Therfore, the proteins are randomly oriented allowing for the entire protein to be availbe for binding. Please contact us to discuss further.

We periodically add new proteins to the array when new full-length cDNA clones are made available.

At present, the amount of each protein on the microarray is not normalized, and varies depending on the protein level expressed in yeast prior to purification. In some rare cases, the protein levels may be low if the expressed protein is toxic to the yeast cells.

Other than controls, the proteins on HuProt are not normalized, however the average protein concentration is ~0.15 mg/mL. Each protein aliquot is printed in duplicate with 100 picoliters (pL) volume per protein spot.

The HuProt microarray contains thousands of membrane proteins. The protein printed on the arrays is eluted using a buffer that contains 0.03% TritonX-100.

We extensively evaluated the folding of many non-membrane proteins on the HuProt microarray (see https://www.cdilabs.com/content/literatures/app-huprot-full-length-folded-3d-proteins). However, we did not perform detailed analysis on the folding of membrane proteins. The full protein content on the HuProt arrays can be found at the top of this page. If you have specific interest in membrane proteins, please consider the VirD™ Membrane Protein Virion Display Microarray which displays proteins embedded in a cell membrane. VirD and HuProt microarrays can be combined in a single service offering from CDI Labs.

All our proteins are eluted in a buffer of pH 8.0–pH8.5 and should work well for studies of post-translational modifications.

Our arrays have been used successfully in a range of different buffers to study serum profiling, protein binding, DNA binding, RNA binding, compound binding, kinase assay, acetylation, SUMOylation/ubiquitinylation, etc. Please contact us to discuss specifics regarding your assay and buffer.

We currently don’t offer test arrays that contain just control spots, however you can practice the steps in your assay with a standard glass microscope slide prior to using the HuProt microarrays. Regarding scanning, the GenePix Pro software has an “auto-PMT” function which can automatically evaluate the required PMT gain values for fast and easy optimization of signal intensity and channel balance.

Please send at least 10 µL of protein–the protein must have a concentration of at least 200 ng/µL. We will dilute the protein to 100 ng/µL in the printing buffer and then print it on the HuProt microarrays ordered. An additional fee is charged for this service. Please allow additional time for these customized arrays to be shipped to you.

We do not recommend stripping as it could affect the protein quality.

Each batch of HuProt microarrays must pass rigorous QC requirements, and comes with a certificate of analysis.

To meet CDI Labs QC standards, >95% of the spots must generate GST signals with a foreground/background ratio >1.5.

Our QC protocol includes sampling at specific points across each print run, to ensure that quality is consistent throughout each printed batch.

The HuProt array contains 29 controls, including fluorophores, as follows:

Control	Function
H1 – Histone H1H2 (A+B) - Histone H2A and H2B mixtureH3 - Histone H3H4 - Histone H4	The histones are non-specific binding proteins used as positive controls for various assays, including serum profiling, antibody specificity assay, protein, DNA, RNA binding assays, etc.
IgG488/594	Alexa Fluor 488/594 labeled IgG, positive control and landmarks for fluorescent detection in 488/594 channels.
Rhodamine + IgG 647	Rhodamine + Alexa Fluor 647 labeled IgG, a positive control and landmarks for fluorescent detection in 532/635 channels.
Anti-human IgA	A positive control for human serum/plasma IgA profiling.
Mouse-anti-biotin	Detects biotin labeled protein probes and serves as a control for anti-mouse antibody detection reagent.
Rabbit-anti-biotin	Detects biotin labeled protein probes and serves as a control for anti-rabbit antibody detection reagent.
BSA - Bovine serum albumin	A negative control for non-specific protein interactions.
Biotin-BSA - biotinylated BSA	A positive control for interaction with streptavidin labeled detection reagent.
Mouse IgM	Positive control for anti-mouse IgM detection.
RanBP2deltaFG	E3 SUMO-Protein Ligase, a positive control for the SUMOylation assay.
hMDM2	E3 Ubiquitin Protein Ligase, a positive control for the ubiquitinylation assay.
Erα	Estrogen receptor alpha. A positive control for ligand binding assays.
Human IgM	A positive control for human serum/plasma IgM profiling.
Human IgG 1.5625 ng/µLHuman IgG 6.25 ng/µLHuman IgG 25 ng/µLHuman IgG 100 ng/µL	The human IgG gradient serves as a positive control for human serum/plasma IgG profiling, and is used for Robust-Linear-Model Normalization in data analysis.
Anti-Human IgG 1.5625 ng/µLAnti-Human IgG 6.25 ng/µLAnti-Human IgG 25 ng/µLAnti-Human IgG 100 ng/µL	The Anti-human IgG gradient serves as a positive control for human serum/plasma IgG profiling, and is used for Robust-Linear-Model Normalization in data analysis.
GST (glutathione S-transferase) 10 ng/µLGST (glutathione S-transferase) 50 ng/µLGST (glutathione S-transferase) 100 ng/µLGST (glutathione S-transferase) 200 ng/µL	The glutathione S-transferase (GST) protein gradient serves as a negative control, and is used for background and statistical significance calculations.
Buffer	Printing buffer only, negative control.

There is one row of control spots at the bottom of each block, including Rhodamine + Alexa Fluor 647 fluorescence as landmarks, but no GFP.

The HuProt microarray is entirely compatible with a range of array scanners. At CDI Labs we employ a GenePix scanner and software for in-house testing services. Yes, we do supply a .gal file for each lot of our arrays, for data generation. It is best to ask the scanner manufacturer if their scanner is compatible with Grace Bio PATH Slides as this is the slides used in HuProt manufacturing.

We analyze scanned array images using GenePix Pro software to get a raw data file (.gpr files). The Z-score of each spot on the array is calculated according to this algorithm: α= Foreground (sample channel), e.g. F635 z = [α – α(avg)]/α(std). α(avg)] and α(std) are the average and standard deviation of α values of all spots on the array. A protein is considered as positive (positive hit) if the average Z-score of its duplicate spots is > 3.

The algorithm above uses the average signal intensity of all spots on the array for normalization.

CDI Labs finds that GenePix Pro is the best software for analysis. We only use the Z-score of the average normalized signal intensity to rank hits.

The average alpha value is of all the spots on a given array.

No. We use the algorithm described above to calculate the average Z- score but not the SNR in the .gpr file.

For protein-DNA interactions the affinity is ~ 500 nM. For protein-protein interactions the affinity can be as low as 1 μM. We have also tested many antibodies with affinities ranging from 500 nM to 0.1 nM. We have not systematically defined the Kd range at which detection drops off.

The HuProt microarray does not include internal standards specific to PPI, but histones are used as positive controls for most applications, including protein-protein interactions. The HuProt microarray is a discovery tool and we typically find dozens, if not hundreds, of positive hits on HuProt – false negatives are not considered a significant issue.

We recommend adding only 0.1 μg/mL of anti-GST to minimize the competition between anti-GST and the mAb (sample) that is being tested. While this will result in weak GST signals, these are strong enough for grid alignment. If you wish to see strong GST signals, you may use 0.5–1.0 μg/mL of anti-GST antibody; however, we have observed a few cases where the secondary antibody cross-reacted with the anti-GST antibody, which resulted in similar signal patterns in the sample channel and the GST channel. If you are familiar with the grid alignment, we encourage you to run the sample mAb only (without anti-GST), or re-probe the array with anti-GST after scanning your antibody signals.

The “n” is ng/μL.

No. The anti-GST information is used to facilitate grid alignment prior to data analysis, but is not used for QC or for normalization of the array data. Our tests using this approach found that many spots with very low GST signals are amplified, so we do not recommend GST normalization for array data analysis (please refer to the list of landmarks above).

The anti-GST antibodies are added only to facilitate grid alignment, therefore only a very low concentration of anti-GST antibody is added (1:10,000 dilution). This minimizes the competition between the anti-GST antibodies and the actual sample being studied. As a result, the GST signals may appear weak or even invisible on some spots. Please note that not all controls contain a GST tag (please refer to the list of landmarks listed).

No, there is no need to strip away the first antibody before re-probing with anti-GST. As long as there are no concerns that the anti-GST could interfere with the primary antibody, you may add both probes simultaneously to the array, and then add the different secondary antibodies simultaneously to detect hits.

GST images are available for each batch printed, and can be downloaded from the CDI Labs website Resources page. (https://cdilabs.com/resources/literature): Typically, we use a 1:2,000 dilution of rabbit anti-GST as the primary antibody, and an Alexa555-anti-Rabbit as the secondary antibody. The blocking buffer is PBS-T with 5% BSA, and the staining protocol used is as stipulated in the users’ manual.

To test mAb specificity, a concentration of 0.1 – 1.0 μg/mL (depending on the mAb affinity) is recommended.

To test the cross-reactivity of your mAb against other proteins on the array, increase the mAb concentration in your assay to 10 μg/mL.

We recommend that each array is used only once for cross-reactivity testing, as the printed proteins will denature during the drying process done prior to scanning. For competition studies, you may add 2 mAbs at the same time to one array (0.1 – 1.0 μg/mL of each mAb).

For analyses using one mAb sample per array, to view the anti-GST staining use a wavelength different from that used to stain your mAb. There are two ways to do this:

1. While the anti-GST antibodies and mAb sample could be added to the array at the same time, CDI Labs does not recommend doing this. Instead,
2. To prevent the anti-GST antibody from competing with your sample mAb or cross-reacting with the secondary antibody, first perform the assay using your sample mAb and then scan the array (most protein spots will not be visible). Next, add the anti-GST antibodies to the array, and then apply the grid pattern to the mAb image. The GST signals will be weak as low concentrations of anti-GST are used. For more information, refer to the user manual above.

CDI Labs has tested the following blood collection tubes (Neat Serum, K2EDTA plasma, K2EDTA plasma, ADC plasma, LiHeparin plasma, NaHeparin plasma) all perform identically in our serum profiling assay.

Please send 20 μL of frozen serum per sample for analysis, preferably on dry ice.

We typically use 1:1000 dilution to ensure that the background is low.

IgG is extremely stable and CDI Labs has successfully tested samples that were stored for > 10 years. It is also best to have minimal freeze thaws.

• HuProt Content: Full list of proteins by name and category.
  https://cdilabs.com/content/literatures/huprot-v40-content


• HuProt User Manual Full list of proteins by name and category.
  https://cdilabs.com/content/literatures/user-manual-huprot-microarray


• HuProt BLAST Nomenclature Full list of proteins by name and category.
  https://cdilabs.com/content/literatures/huprot-blast-nomenclature


• Human Protein Atlas Overlap to HuProt Full list of proteins by name and category.
  https://cdilabs.com/content/literatures/huprot-hpa-alignment