The SARS-CoV-2 proteome array described above was assembled in an incubation tray and then blocked with 5% (w/v) milk in 1× PBS with 0.05% (v/v) Tween-20 (T) for 10 min at room temperature. After washing with PBST three times, the resulting array was incubated with 100-fold diluted serum in 5% (w/v) milk in PBST for 30 min at room temperature with gentle shaking. After washing again, the array was then incubated for 30 min with a mixture containing Cy3 Affinipure donkey anti-human IgG (H + L) and Alexa fluor 647 Affinipure goat anti-human IgM FC5µ antibody (Jackson ImmunoResearch, USA) (2 μg/mL). Finally, the array was washed with PBST buffer, dissembled from the tray, and dried via centrifugation for 2 min at 2000 rpm. The array was scanned with a GenePix 4300 A microarray scanner (Molecular Devices, Sunnyvale, CA, USA), and signals extracted using GenePix Pro7 software (Molecular Devices, Sunnyvale, CA, USA). These experiments were performed in a biosafety level 3 (BSL3) laboratory.
Genepix 4300a microarray scanner
Manufactured by Molecular Devices
Sourced in United States
The GenePix 4300A is a microarray scanner designed for high-quality imaging of DNA, protein, and other samples. It features a dual-laser system that enables the detection of multiple fluorescent dyes, and its high-resolution optics provide sharp, detailed scans. The GenePix 4300A is capable of rapid, high-throughput scanning to support efficient analysis of microarray data.
Automatically generated - may contain errors
Lab products found in correlation
Genepix pro 7, by Molecular Devices (6 mentions)
Cy3 affinipure donkey anti human igg h l, by Jackson ImmunoResearch (2 mentions)
Alexa fluor 647 affinipure goat anti human igm, by Jackson ImmunoResearch (1 mentions)
Alexafluor 647 goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Rabbit igg fraction, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg cy5 conjugate, by Merck Group (1 mentions)
11 protocols using genepix 4300a microarray scanner
1
SARS-CoV-2 Proteome Array Profiling
2
Lectin and Antibody Microarray Analysis
3
SARS-CoV-2 S Protein Peptide Microarray
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The peptide microarray containing eight S proteins and tiled peptides representing the SARS-CoV-2 S protein was prepared as described in our previous work 19 (link), 40 (link). The array was assembled in an incubation tray and blocked with 5% (w/v) milk in phosphate-buffered saline (PBS) containing 0.05% (v/v) Tween-20 (PBST) for 30 min at room temperature before antibody detection. After aspirating the blocking solution, the 1:200 diluted serum was added to the array and incubated at room temperature for 20 min. After washing three times with PBST, the array was then incubated for 20 min with a Cy3 labelled donkey anti-human IgG(H+L) antibody (Jackson ImmunoResearch, USA) (2 μg/mL). Finally, the array was washed with PBST and deionized water, disassembled from the tray and dried with vacuum pump. The slide was scanned at 532 nm using a GenePix 4300A microarray scanner (Molecular Devices, Sunnyvale, CA, USA). The median fluorescent signal intensity of each spot with background subtraction was extracted using GenePix Pro7 software (Molecular Devices, Sunnyvale, CA, USA). The raw data for each peptide of the S protein were normalized to the z-score. The immunogenic peptides were defined as those with a z-score >1.96. Epitope identification via epitope mapping was performed as described in our previous work 60 (link).
4
Peptide Microarray Immunoassay Protocol
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The peptide microarrays were assembled in an incubation tray and blocked with 5% (weight‐bulk ratio) milk/(PBS with 0.2% [volume ratio] Tween‐20 or PBST) for 1 min at room temperature. After being washed three times with PBST, the array was incubated with plasma at a dilution of 1/300 for 30 min at room temperature. The microarray was then incubated for 30 min with a mixture containing Cy3 AffiniPure donkey anti‐human IgG (H+L) and Alexa Fluor 647 AffiniPure goat anti‐human IgM FC5 μ antibody (2 μg/mL; both from Jackson ImmunoResearch). Finally, the array was washed with PBST and water, dissembled from the tray, and dried using centrifugation for 2 min at 2000 rpm. The array was scanned with a GenePix 4300A microarray scanner (Molecular Devices) at 10 μm resolution using a laser at 532 nm with 100% power/PMT Gain 800 for IgG. The median fluorescent signal intensity with background subtraction was extracted using GenePix Pro7 software (Molecular Devices).
5
SARS-CoV-2 Spike Variant Antibody Assay
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Prior to antibody detection, the SARS-CoV-2 spike variant protein microarrays were assembled in an incubation tray and blocked with 1% (w/v) milk in 1x phosphate buffered saline, pH 7.2 (PBS), with 0.2% (v/v) Tween-20 (PBST) for 10 min at room temperature. After washing with PBST three times, the array was incubated with serum diluted 10- to 320-fold in PBST for 30 min or diluted antibodies for 1 h at room temperature. After washing again, the array was then incubated for 60 min with Cy5-labeled ACE2 (50 ng/mL). Finally, the array was washed with PBST and water, dissembled from the tray, and dried via centrifugation for 2 min at 2,000 rpm. The array was scanned with a GenePix 4300A microarray scanner (Molecular Devices, Sunnyvale, CA, USA) at a 10 μm resolution using a laser at 635 nm with 100% power/ PMT Gain 900. The median fluorescent signal intensity with background subtraction was extracted using GenePix Pro7 software (Molecular Devices, Sunnyvale, CA, USA). The neutralizing antibody titer of serum is expressed by the effective concentration (EC50). EC50 is the dilution of sera that inhibits 50% S-ACE2 binding in the mSAIS assay. EC50 was calculated using the GraphPad Prism software 8.3 based on the fluorescent signal intensity.
6
Microarray Analysis of miR-206 Overexpression
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Undifferentiated HC11 cells were transfected with miR‐206 mimic or negative control, twice in 24 hr intervals, and microarray analysis was performed 24 hr after final transfection. RNA isolated after treatments were analyzed in biological and technical duplicates. Spotted 70‐mer arrays covering 36,000 genes and variants (full protein‐coding genome) were used (Human Genome OpArray, Microarray Inc., Huntsville, AL) as previously described (Edvardsson, Ström, Jonsson, Gustafsson, & Williams, 2011 ; Simon, Mesmar, Helguero, & Williams, 2017 ). Slides were hybridized using a dye‐swap design and scanned using GenePix 4300A microarray scanner (Molecular Devices, Sunnyvale, CA).
7
Profiling SARS-CoV-2 Antibody Responses Using Proteome Microarray
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The proteome microarrays containing full-length N, full-length E, and truncated S proteins of SARS-CoV-2 and 966 peptides representing SARS-CoV-2 proteins were prepared as described previously [17] . The proteome microarrays were assembled in an incubation tray and blocked with 5% (w/v) milk in PBS containing 0.05% (v/v) Tween-20 (PBST) for 10 min at room temperature before antibody detection. After aspirating the blocking solution, 1:101 diluted serum was added to the array and incubated at room temperature for 30 min. After washing three times with PBST, the array was then incubated for 20 min with a mixture containing Alexa Fluor ®647 Affinipure goat anti-human IgM(Jackson ImmunoResearch, USA, CAT#709–165-149) and Cy™3 Affinipure donkey anti-human IgG(H + L) antibody(Jackson ImmunoResearch, USA, CAT#109–605-043) (4 μg/mL) or only Cy™3 AffiniPure goat anti-Human serum IgA antibody(Jackson ImmunoResearch, USA, CAT#109–165-011) (4 μg/mL). Finally, the array was washed with PBST and deionized water, dissembled from the tray, and dried with a vacuum pump. The proteome microarray was scanned at 532 and 635 nm using a GenePix 4300A microarray scanner(Molecular Devices). The median of fluorescent signal intensity with the deduction of the background was extracted using GenePix Pro7 software.
8
SARS-CoV-1 N-Protein Antibody Screening
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The peptide microarrays were assembled in an incubation
tray and blocked with 5% (w/v) milk in 1X PBS with 0.2% (v/v) Tween-20
(PBST) for 1 min at room temperature. After it was washed with PBST
three times, the array was incubated with a rabbit anti-SARS-CoV-1
N-protein monoclonal or a rabbit anti-SARS-CoV-1 N-protein polyclonal
antibody (1 μg/mL) (catalogue Nos. 40143-R001 and 40143-T62,
respectively; Sino Biological) for 30 min at room temperature. After
it was washed again, the array was incubated with an Alexa Fluor 555
labeled goat antirabbit IgG (H+L), cross-adsorbed, secondary antibody
(Jackson ImmunoResearch) for 30 min. The arrays were washed, dissembled
from the tray, and dried with centrifugation for 2 min at 2000 rpm.
The resulting array was scanned with a GenePix 4300A microarray scanner
(Molecular Devices). The median fluorescent signal intensity of each
spot was extracted using GenePix Pro7 software (Molecular Devices).
The median background signal was subtracted from the median spot signal
intensity.
tray and blocked with 5% (w/v) milk in 1X PBS with 0.2% (v/v) Tween-20
(PBST) for 1 min at room temperature. After it was washed with PBST
three times, the array was incubated with a rabbit anti-SARS-CoV-1
N-protein monoclonal or a rabbit anti-SARS-CoV-1 N-protein polyclonal
antibody (1 μg/mL) (catalogue Nos. 40143-R001 and 40143-T62,
respectively; Sino Biological) for 30 min at room temperature. After
it was washed again, the array was incubated with an Alexa Fluor 555
labeled goat antirabbit IgG (H+L), cross-adsorbed, secondary antibody
(Jackson ImmunoResearch) for 30 min. The arrays were washed, dissembled
from the tray, and dried with centrifugation for 2 min at 2000 rpm.
The resulting array was scanned with a GenePix 4300A microarray scanner
(Molecular Devices). The median fluorescent signal intensity of each
spot was extracted using GenePix Pro7 software (Molecular Devices).
The median background signal was subtracted from the median spot signal
intensity.
9
Microarray Immunoassay for Antibody Profiling
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Microarray slides were blocked with 1% BSA in PBS (w/v) for 1 h at RT, washed three times with PBS and dried by centrifugation (300 g, 5 min). FlexWell 64 incubation chambers (Grace Bio-Labs) were applied to microarray slides. Slides were incubated with mouse sera diluted 1:100 with PBS unless mentioned otherwise, in a humid chamber for 1 h at RT, washed three times with 0.1% Tween-20 in PBS (v/v) and dried by centrifugation (300 g, 5 min). Slides were incubated with fluorescence-labelled secondary antibodies diluted 1:400 in 1% BSA in PBS (w/v) in a humid chamber for 1 h at RT, washed three times with 0.1% Tween-20 in PBS (v/v), rinsed once with deionized water and dried by centrifugation (300 g, 5 min) before scanning with a GenePix 4300A microarray scanner (Molecular Devices). Image analysis was carried out with the GenePix Pro 7 software (Molecular Devices). The photomultiplier tube voltage was adjusted such that scans were free of saturation signals. Background-subtracted mean fluorescence intensity values were exported to Microsoft Excel, for further analyses. Secondary antibodies used were as follows: Alexa Fluor 647 Goat Anti-Mouse IgG (H+L) (Life Technologies, catalogue number A-31574) and Alexa Fluor 594 Goat Anti-Mouse IgG1 (γ1) (Life Technologies, catalogue number A-21125).
10
Chemical Array Screening of Plant Proteins
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The slides of the RIKEN NPDepo (Osada, 2010 (link)) were prepared and analyzed as previously published (Bürger et al., 2012 (link); Hagiwara et al., 2010 (link); Kanoh et al., 2006 (link); Kondoh et al., 2015 (link); Miyazaki et al., 2008 (link); Zimmermann et al., 2013 (link)). Chemical array screening was performed in 20 mM HEPES, 150 mM NaCl, final pH 7.53–7.55. For protein and antibody incubation, the array slide was covered by a gap cover glass (24 × 60 mm) from Matsunami Glass Ind., Ltd and incubated using 50 μl of GST‐AtDLK2, GST‐AtSOBER1, or GST‐AtTIPSY1 solution (1 μM) in the above buffer containing 1% skim milk at 30°C for 1 h. After washing, array slides were incubated with anti‐GST antibody (rabbit IgG fraction, Invitrogen, 30 μg/ml) in the same buffer containing 1% skim milk at 30°C for 1 h. This incubation was followed by another wash step and incubation with a second antibody (Millipore, goat anti‐rabbit IgG, Cy5 conjugate, 50 μg/ml) at 30°C for 1 h. After the final wash step, slides were scanned at 635 nm on a GenePix 4300A microarray scanner (Molecular Devices).
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