Recombinant human IgG1 mAbs were screened for binding on protein microarrays (ProtoArray) (catalog no. PAH0525101; Invitrogen) precoated with >9,400 human proteins in duplicate. The binding patterns of human anti-HA bnAbs were compared to the human myeloma protein 151 K in lot-matched arrays. Array-bound anti-human IgG served as the loading control for the detection Ab, and array-bound human IgG served as the loading control for the secondary reagent. Abs were screened for reactive antigens on protein microarrays following the manufacturer’s instructions and as described previously16 (link). The ProtoArray microarray (Invitrogen) was blocked and incubated on ice with 2 μg/ml of HA mAb or isotype control 151 K for 90 min. Ab binding to array protein was detected with 1 μg/ml of Alexa Fluor 647-labeled anti-human IgG secondary Ab (Invitrogen). The ProtoArray microarrays were scanned using a GenePix 4000B scanner (Molecular Devices) at 635 nm, with 10-μm resolution. Fluorescence intensities were quantified with GenePix Pro 5.0 program (Molecular Devices) using lot-specific protein location information provided by the microarray manufacturer.
Protoarray
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ProtoArray is a high-throughput protein microarray platform designed for the identification and characterization of protein-protein, protein-small molecule, and protein-antibody interactions. The ProtoArray contains over 9,000 unique human proteins expressed in a baculovirus system, providing a comprehensive platform for studying a wide range of protein interactions.
Automatically generated - may contain errors
Lab products found in correlation
Genepix pro 5.0 program, by Molecular Devices (3 mentions)
Genepix 4000b scanner, by Molecular Devices (3 mentions)
E3 substrate id, by LifeSensors (2 mentions)
Axon scanner, by Thermo Fisher Scientific (2 mentions)
Prospector free software, by Thermo Fisher Scientific (2 mentions)
Genepix 4000b, by Molecular Devices (1 mentions)
Streptavidin alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Human gene st 2.1 microarrays, by Thermo Fisher Scientific (1 mentions)
19 protocols using protoarray
1
Screening Recombinant Human IgG1 mAbs
2
High-Throughput Protein Microarray Binding
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Binding of recombinant CH103 Ab mutants to 9,400 human proteins was determined using a ProtoArray (Invitrogen, Waltham, MA) in duplicate, according to the manufacturer instructions and as described previously (13 (link), 23 (link), 50 (link), 51 (link)). In brief, protein microarrays were blocked with 2 µg/ml of recombinant Abs or 151K, an isotype-matched (IgG1, κ) human myeloma control Ab (Southern Biotech) for 1.5 hours at 4°C. Ab binding to array proteins was detected using 1 µg/ml of Alexa Fluor 647-conjugated anti-human IgG (Invitrogen), using mild agitation at 4°C for 1.5 hours. Microarrays were scanned using a GenePix 4000B scanner (Molecular Devices, San Jose, CA). Fluorescence intensities of Ab binding to each protein were quantified by aligning image data with the GenePix Pro 5.0 program (Molecular Devices) using lot-specific protein spot definitions provided by the manufacturer, and by comparing Ab-binding patterns to the 151K control.
3
Protein Microarray Analysis of SARS-CoV-2 Protein Reactivity
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Reactivity of recombinant S proteins from Wuhan and VOC Omicron as well as of recombinant RBD (Wuhan, Delta, and Omicron) to large set (>9,000) of human proteins was assessed by using protein microarrays (ProtoArray®, Invitrogen, Thermo Fischer). First microarray chips were blocked by incubation for 1 hr at 4°C with buffer containing: 50 mM HEPES, pH 7.5; 200 mM NaCl; 0.08% Triton‐X‐100; 20 mM reduced glutathione; 1 mM dithiothreitol; 25% glycerol, and 1× synthetic block (Thermo Fischer). After washing 2 × 5 min with PBS containing 0.1% Tween 20, the chips were incubated for 90 min at 4°C with S proteins and RBDs labeled with Alexa Fluor™ 555, diluted to 4.5 and 1 μg/ml, respectively in PBS containing 0.1% Tween 20. After washing 2 × 5 min with PBS containing 0.1% Tween 20, chips were soaked in deionized water, dried by centrifugation (200 × g) for 1 min. The fluorescence intensity was measured by using microarray scanner GenePix 4000B (Molecular Devices, San Jose, CA). The microarray chips were analyzed by using Spotxel software v. 1.7.7 (Sicasys, Heidelberg, Germany). The Z‐scores for reactivity of SARS CoV‐2 proteins to each human protein were determined by using ProtoArray Prospector v 5.2 software (Invitrogen, Thermo Fisher).
4
Screening Monoclonal Antibodies on Protein Microarray
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MAbs were screened for binding on protein microarrays (ProtoArray) (PAH0525101; Invitrogen) pre-coated with 9,400 human proteins in duplicate and screened following manufacturer’s instructions and as previously described (Liu et al., 2015 (link)). Briefly, after blocking, the microarray was incubated on ice with 2 μg/ml of mAbs or isotype control 151K for 90 min. Ab binding to array protein was detected with 1 μg/ml of Alexa Fluor 647-labeled anti-human IgG (Invitrogen) secondary Ab. Microarrays were scanned using a GenePix 4000B scanner (Molecular Devices) at a wavelength of 635 nm, with 10-μm resolution, using 100% power and 600 gain. Fluorescence intensities were quantified with GenePix Pro 5.0 program (Molecular Devices) using lot-specific protein location information provided by the microarray manufacturer.
5
Screening Monoclonal Antibodies on Protein Microarrays
6
Protein Microarray Protocol for GST-tagged Proteins
7
Comprehensive Analysis of LIMS1 in Kidney Disease
8
Ubiquitination Validation of Regulatory Proteins
9
Identifying ROCO Protein Interactors
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3xFLAG tagged, full‐length DAPK1, LRRK1, LRRK2, MASL1, and GFP control proteins were purified as previously described.32 Six micrograms of each purified 3xFLAG tagged protein were used to probe protein microarrays (Protoarray, version 4.1; Invitrogen) according to the manufacturer's instructions with the modification that after 3xFLAG tagged protein probing, arrays were probed with monoclonal ANTI‐FLAG BioM2−Biotin, Clone M2 (Sigma‐Aldrich) antibody, followed by probing with Alexa Fluor 647 streptavidin (Invitrogen).33 Arrays were imaged using an Axon GenePix 4000B fluorescence scanner and images were analyzed using GenePix Pro software. Protoarray Prospector software was used to analyze the microarray data acquired from GenePix Pro and identify the significant hits. Binding strength was estimated as Z‐scores, that is, numbers of standard deviations above background fluorescence on the array. Each protein on the array was spotted in duplicate, hence reported values were averaged for both spots. Signals considered as potential interactions were determining using a Z‐score threshold of Z > 3. ROCO protein positive hit interactors were determined by filtering against GFP (negative control) interactions to identify proteins that bound DAPK1, LRRK1, LRRK2, or MASL1 but not GFP.
10
Ubiquitination Validation of Regulatory Proteins
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Validation of hnRNPA1, TAK1, and Tubulin ubiquitination was performed in accordance with the manufacturer recommendations (E3 Substrate ID, LifeSensors, Inc). In vitro ubiquitin reconstitution assays were performed on two protein arrays (Invitrogen ProtoArray). Recombinant E1 (UBE1), E2 (UBE2N/UBE2V1), wild-type ubiquitin, and ATP were included in each reaction. One array also received a mix containing purified human TRAF6 protein. Arrays were visualized with Tandem Ubiquitin Binding Entities (TUBEs) and FK2 followed by the corresponding Cy-dye labeled secondary detection reagents. To calculate ubiquitination of hnRNPA1, TAK1, and Tubulin, median intensities subtracted from background intensities were utilized from biological replicates (F532 Median - B532 Median = ubiquitin intensity).
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