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Protoarray human protein microarray v5

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The ProtoArray® Human Protein Microarray v5.0 is a high-density protein microarray that contains more than 9,400 human proteins expressed in insect cells. The array is designed to enable the study of protein-protein interactions, protein-small molecule interactions, and protein-lipid interactions.

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Lab products found in correlation

Anti v5 alexa fluor 647 antibody, by Thermo Fisher Scientific (1 mentions) Prospector software, by Thermo Fisher Scientific (1 mentions) Alexa fluor 647 conjugated streptavidin, by Thermo Fisher Scientific (1 mentions) Alexa 647, by Thermo Fisher Scientific (1 mentions) Genepix 4200al, by Molecular Devices (1 mentions) Protoarray prospector software, by Thermo Fisher Scientific (1 mentions) Pcr2.1 topo vector, by Thermo Fisher Scientific (1 mentions) In vitro transcription, by Promega (1 mentions) Geneart, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

ProtoArray (19 citations) Protoarray® v5.0 (10 citations) ProtoArray® Human Protein Microarrays v5.0 (4 citations) Human ProtoArray v5.0 (2 citations)

7 protocols using protoarray human protein microarray v5

1

Purification and Microarray Analysis of HCV NS5A Protein

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HCV NS5A protein expressed from Escherichia coli was purified using Invitrogen Ni-nitrilotriacetic acid (Ni-NTA) agarose beads according to the manufacturer’s instructions. Firstly, ProtoArray® Human Protein Microarray v5.0 (Invitrogen) was incubated with blocking buffer (50 mM HEPES [pH 7.5], 25% glycerol, 0.08% Triton X-100, 200 mM NaCl, 20 mM reduced glutathione, and 0.1 mM dithiothreitol [DTT]) for 1 h at 4°C. Next, purified NS5A protein diluted in probing buffer (phosphate-buffered saline [PBS] containing 0.1% Tween 20) was added to the protein microarray. Following incubation at 4°C for 1.5 h, the array was washed five times in ice-cold buffer and treated with Anti-V5-Alexa Fluor 647 antibody (Invitrogen) for 1.5 h at 4°C. The images were scanned using a PerkinElmer ScanArray Ex-press HT system and analyzed by the Invitrogen Prospector software (ver. 5.2).
Lim Y.S., Nguyen M.T., Pham T.X., Huynh T.T., Park E.M., Choi D.H., Kang S.M., Tark D, & Hwang S.B. (2021). Hepatitis C Virus Nonstructural 5A Protein Interacts with Telomere Length Regulation Protein: Implications for Telomere Shortening in Patients Infected with HCV. Molecules and Cells, 45(3), 148-157.
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2

Protein Microarray Assay for Immune Profiling

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The Invitrogen ProtoArray Human Protein Microarray v5.0 (9,480 human proteins) assay and statistical analyses were performed by UT Southwestern Genomics and Microarray Core Facility. In short, protein microarrays were blocked with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 0.1% Tween-20 before incubation with serum (1:500) and Cy3-conjugated anti-human IgM and Alexa Fluor 647-conjugated anti-human IgG. The arrays were scanned using an Axon Genepix 400B fluorescent microarray scanner. Genepix 6.0 software was used to align the scanned image to the template and to determine the pixel intensities for each spot on the array. The reported pixel intensity was calculated as the average of duplicate signals after background subtraction. The software tool used was the Prospector software (Invitrogen), which is based on M-statistics. This software performs background subtraction, normalization of the signals, and analysis of the differences between the two groups of patients. When comparing two groups, a cutoff for positivity is calculated for each protein using M-statistics. The proportion of subjects with an immune response above the cutoff value is counted and a P-value representing the significance of the difference between both groups is calculated.
Luo Y., Brigham D., Bednarek J., Torres R., Wang D., Ahmad S, & Mack C.L. (2021). Unique cholangiocyte-targeted IgM autoantibodies correlate with poor outcome in biliary atresia. Hepatology (Baltimore, Md.), 73(5), 1855-1867.
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3

Protein Microarray for Novel Antigen Discovery

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A Protoarray Human Protein Microarray v5.0 (Invitrogen Life Technologies) with over 9000 purified human proteins was used for novel antigen discovery. The purified recombinant proteins spotted on nitrocellulose-coated glass slides were reacted with the serum of SLE subjects or healthy controls and subsequently incubated with biotinylated goat anti-human IgE antibody (5 µg/ml) (Vector Laboratories) to detect IgE antibody binding. Then either an AlexaFluor 647-conjugated streptavidin (Invitrogen, 1 µg/ml) or an AlexaFluor 647-conjugated anti-V5 antibody (Invitrogen, 0.1 µg/ml) was added for detection and normalization, respectively. Arrays were scanned using fluorescent microarray GenePix 4000B Scanner and GenePix 6.0 software was used for data acquisition. Data analysis was done using Invitrogens proprietary Protoarray Prospector software. For each protein spotted in every array, Z-Score, Z-factor, CI-P value (Chebyshevs Inequality p-Value) was measured. M-Statistic is used to identify those proteins which show a significant differential signal between two populations. The “cutoff” value used corresponded to the value of 200 RFU above the M-Statistic signal threshold established for a specific protein.
Dema B., Pellefigues C., Hasni S., Gault N., Jiang C., Ricks T.K., Bonelli M.M., Scheffel J., Sacré K., Jablonski M., Gobert D., Papo T., Daugas E., Illei G., Charles N, & Rivera J. (2014). Autoreactive IgE Is Prevalent in Systemic Lupus Erythematosus and Is Associated with Increased Disease Activity and Nephritis. PLoS ONE, 9(2), e90424.
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4

Biotinylated Calmodulin Protein Microarray Assay

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Wild-type and M77Q calmodulins were biotinylated with maleimide-PEG2-Biotin (Invitrogen, # 21901) and then dialyzed 50 mM HEPES, 150 mM NaCl and 1 mM DTPA. The expected increase in mass of 525.6 Da was confirmed by HPLC-time of flight mass spectroscopy. The concentrations, ~300 µM were measured after which an aliquot was diluted to 0.10 µM in buffer I (50 mM HEPES, 150 mM NaCl, 5% glycerol, 0.05% TritonX-100, 1% bovine serum albumin, 5 mM MgCl2, 0.5 mM dithiothreitol (DTT) and 2 mM CaCl2). The solution was incubated with 0.2 µg/mL streptavidin-conjugated Alexa 647 (Invitrogen S32357) in the dark at room temperature for 1 h. Protein arrays (ProtoArray® Human Protein Microarray v. 5.0, Invitrogen) were incubated with blocking buffer (50 mM HEPES, 150 mM NaCl, 0.1% Tween-20 and 1% bovine serum albumin (BSA)) for 1 h at 4° and then incubated with the biotin-streptavidin complex for 2 h at 4°. Arrays were then washed 3 times with washing buffer II (50 mM HEPES, 150 mM NaCl, 0.1% Tween-20, 1% BSA, 5 mM MgCl2 and 0.5 mM dithiothreitol [DTT]). Arrays were scanned with a GenePix 4200AL (Molecular Devices, San Jose, CA, USA) and analyzed with Invitrogen’s ProtoArray Prospector software (Thermo Fisher Scientific, Waltham, MA, USA).
Marimoutou M., Springer D.A., Liu C., Kim G, & Levine R.L. (2018). Oxidation of Methionine 77 in Calmodulin Alters Mouse Growth and Behavior. Antioxidants, 7(10), 140.
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5

In vitro RNA Probing on Protein Microarray

Cited 1 time
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In vitro RNA production and labeling followed by probing the ProtoArray Human Protein Microarray v5.0 (Life Technologies cat# PAH0525101) were performed as described (Siprashvili et al., 2012 (link)).
Xue Z., Hennelly S., Doyle B., Gulati A.A., Novikova I.V., Sanbonmatsu K.Y, & Boyer L.A. (2016). A G-rich motif in the lncRNA Braveheart interacts with a zinc finger transcription factor to specify the cardiovascular lineage. Molecular cell, 64(1), 37-50.
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6

Profiling RNA-Protein Interactions

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DNA encoding SNORD50A and SNORD50B was synthesized by overlapping PCR of a construct with an SP6 promoter at the 5′ end, the resulting fragment was cloned into the pCR2.1TOPO vector (Life Technologies), and RNAs were produced and labeled with Cy5 dye with a ratio of RNA:Label IT Cy5 reagent of 1:3 and a reaction time of not more then 30 min at 37 °C to achieve between 0.5 and 1 Cy5 dye unit covalently attached to each RNA used. For RNA incubation, the ProtoArray Human Protein Microarray v5.0 (Life Technologies) was used. GO analysis and PFAM domain analysis of RNA-binding proteins were performed with Benjamini-Hochberg correction of the P value. The P value for the Venn diagram illustrating overlap of two independent microarray incubations was calculated using Fisher’s exact test.
Siprashvili Z., Webster D.E., Johnston D., Shenoy R.M., Ungewickell A.J., Bhaduri A., Flockhart R., Zarnegar B.J., Che Y., Meschi F., Puglisi J.D, & Khavari P.A. (2015). The noncoding RNAs SNORD50A and SNORD50B bind K-Ras and are recurrently deleted in human cancer. Nature genetics, 48(1), 53-58.
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7

Identification of SLINKY RNA Interactors

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DNAs encoding SLINKY transcript variants 1 and 2 were generated by GeneArt gene synthesis (Thermo Fisher Scientific ), and subcloned into pBluescriptKS. SLINKY RNAs were then synthesized by in vitro transcription (Promega), and labeled with Cy5 (Label IT μArray labeling kit; Mirus) to achieve approximately 1 to 3 Cy5 fluorescence dye per transcript. Labeled RNAs were then hybridized to the ProtoArray Human Protein Microarray v5.0 (Life Technologies), and signal scanned and quantified as previously described [20 (link)]. Promiscuous RNA binding proteins, those that have bound to more than 75% of all labelled RNAs (over 50) that we have collectively assayed (ref [20 (link)] and unpublished), were excluded from analysis.
Gong X., Siprashvili Z., Eminaga O., Shen Z., Sato Y., Kume H., Homma Y., Ogawa S., Khavari P.A., Pollack J.R, & Brooks J.D. (2017). Novel lincRNA SLINKY is a prognostic biomarker in kidney cancer. Oncotarget, 8(12), 18657-18669.
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