Protein expression screening by the Label-based Mouse Antibody Array 1-the differential screening antibody microarray, which was designed and manufactured by RayBiotech, Inc. (Norcross, USA), contains 308 antibodies [33] (link). Retina protein obtained from db/db or db/m mice were pretreated and resultant supernatant was collected with measured protein contents. Each of the antibodies has two replicates that are printed on a coated glass microscope slide, along with multiple positive and negative controls. The antibody array experiment was performed by Wayen biotechnologies (Shanghai), according to the established protocol. Fluorescence Detection were scanned (GenePix 4000B, Axon Instruments, USA) until glass chips were completely dry and the chips were scanned on a GenePix ×4000 scanner (GenePix 4000B, Axon Instruments, USA) and the images were analyzed with GenePix Pro 6.0 (Axon Inxtruments, USA). After subtracting background signals and normalization to positive controls, comparison of signal intensities between and among array images can be used to determine relative differences in expression levels of each protein between groups. Any ≥1.5-fold increase or ≤0.65-fold decrease in signal intensity for a single analyte between samples or groups may be considered a measurable and significant difference in expression, provided that both sets of signals are well above background.
Genepix 4000b
Manufactured by Molecular Devices
Sourced in United States, Denmark
The GenePix 4000B is a microarray scanner designed for high-throughput analysis of gene expression and protein arrays. It features a dual-laser excitation system and high-resolution imaging capabilities to accurately detect and quantify fluorescent signals on microarray slides.
Automatically generated - may contain errors
Lab products found in correlation
Genepix pro 6, by Molecular Devices (17 mentions)
Array pro image analysis software, by Media Cybernetics (8 mentions)
Genepix pro, by Molecular Devices (5 mentions)
Antibody array kit, by Full Moon BioSystems (5 mentions)
Phosphorylation proarray, by Full Moon BioSystems (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Cyanine3 (cy3), by GE Healthcare (4 mentions)
Streptavidin cy5, by Thermo Fisher Scientific (4 mentions)
Genepix 3, by Molecular Devices (3 mentions)
Antibody array assay kit, by Full Moon BioSystems (3 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (3 mentions)
Array pro analyzer, by Media Cybernetics (2 mentions)
Genepix pro 3, by Molecular Devices (2 mentions)
Mouse cytokine array g3, by RayBiotech (2 mentions)
Genepix 6, by Molecular Devices (2 mentions)
Amino allyl messageamp arna kit, by Thermo Fisher Scientific (2 mentions)
L series human antibody array 507, by RayBiotech (2 mentions)
Hs 4800 pro system, by Tecan (2 mentions)
Animo allyl messageamp 2 arna amplifi cation kit, by Thermo Fisher Scientific (2 mentions)
Isogen, by Nippon Gene (2 mentions)
157 protocols using genepix 4000b
1
Differential Protein Expression Analysis Using Mouse Antibody Array
2
Proteomic Profiling of UCB-MSCs and NR8383 Cells
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We prepared conditioned media from each group (NR8383 cells alone or LPS-treated NR8383 cells; UCB-MSCs alone or UCB-MSCs with LPS-treated NR8383 cells). Protein expression screening was conducted using the Label-Based Human Antibody Array, a differential screening antibody microarray (RayBiotech, Peachtree Corners, GA, USA) which contains 507 antibodies (Supplementary Table 2 ). The antibody array experiment was performed by E-biogen (Seoul, Korea), according to an established protocol. Fluorescence detection were conducted using a GenePix 4000B (Axon Instruments, Union City, CA, USA) until the glass chips were completely dry, and the chips were scanned on a GenePix ×4000 scanner (GenePix 4000B, Axon Instruments) and the images were analyzed with GenePix Pro 6.0 software (Axon Instruments). After subtracting the background signals and normalizing the values to the positive controls, signal intensities between and among array images were compared to determine the relative differences in expression levels of each protein between groups (UCB-MSCs alone vs. UCB-MSCs with LPS-treated NR8383 cells).
3
Glycan Microarray Analysis of Lectin Interactions
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Thirty seven lectins with different binding preferences covering N- and O-linked glycans were spotted on homemade epoxysilane-coated slides. Each lectin was spotted in triplicate per block, with quadruplicate blocks on one slide. After immobilization, the slides were blocked with blocking buffer containing 2% BSA in 1 × PBS (0.01 mol/L phosphate buffer containing 0.15 mol/L NaCl, pH 7.4) for 1 h, rinsed twice with 1 × PBST (0.2% Tween 20 in 1 × PBS) for 5 min each, and finally rinsed in 1 × PBS before drying. The microarrays were scanned using a Genepix 4000B confocal scanner (Axon Instruments, Foster City, CA, USA) set at 70% photomultiplier tube and 100% laser power. The acquired images were analyzed at 532 nm for Cy3 detection by the Genepix 3.0 software (Version 3, Axon Instruments Inc., Union City, CA, USA).
4
Microarray Data Analysis Protocol
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Hybridized microarrays were scanned (photomultiplier tube, 500–700; pixel size, 5; focus position, 130) using a GenePix 4000B microarray scanner (Axon Instruments, Union City, CA, USA). After data extraction (https://webapps.combimatrix.com ), the background was calculated for individual samples using factory-built controls with low intensities (lowest intensity, 5–30%) and the median signal intensities were calculated for subtraction. Microarray data of individual samples were normalized by global normalization using probes with signal values greater than zero, < 60,000 (the saturation value), and greater than the lowest 5% of the signal value of each sample. In total, 3,382 probes were used for the final analysis. The local-pooled-error (LPE) test (http://bioinformatics.oxfordjournals.org/cgi/reprint/19/15/1945 ) and fold-change analysis were applied to determine differentially expressed sets of genes using the Avadis Prophetic software version 3.3 (Strand Genomics Ltd., Bangalore, India). For further analysis of the upregulated and downregulated genes, gene ontology (GO) term annotation (biological process, molecular function, and cellular component) and enrichment analysis were performed using the Blast2GO software with default parameters (Conesa et al., 2005 (link)).
5
Microarray Analysis of Mycobacterium tuberculosis
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Mtb oligoarray slides were kindly provided by Dr. Stefan H. E. Kaufmann (Max Planck Institute for Infection Biology, Berlin, Germany). RNA labelling and array hybridisation were performed as described previously59 (link). The hybridised slides were scanned using a GenePix 4000B microarray scanner (Axon Instruments, CA, USA), and spot intensities were identified and quantified with the TM4 Microarray Software Suite (http://www.tm4.org ). The spot signal intensities were normalised in MIDAS using the LOWESS algorithm options and total array intensity. Four biological replicate arrays for exponential-growth condition and three biological replicates for hypoxic-culture condition were used for statistical analysis.
6
Yeast pre-mRNA Microarray Analysis
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The following is adapted from previously designed S. cerevisiae pre-mRNA microarray studies (Pleiss et al. 2007 (link)). Briefly, 100 mL of SAD1 pRS313/sad1▵::kan and sad1-1 pRS313/sad1▵::kan cells were grown to mid-log phase at 25°C and then split to two 50 mL cultures, which were either kept at 25°C or shifted to 37°C for 30 min until cells were collected and frozen. RNA was extracted and split for dN9 cDNA synthesis in the presence of either Cy5- or Cy3-labeled dUTP. Cy3/Cy5-labeled SAD1 and sad1-1 cDNAs were competitively hybridized to pre-mRNA microarrays composed of probes that were designed to hybridize to sequence elements in the intron, the exon, and the junction of every yeast intron-containing gene. Microarray fluorescence was measured at 532 and 635 nm with an Axon Instruments GenePix 4000B, and images were processed with Axon Instruments GenePix Pro, version 5.1. Six technical replicate spots per array and dye flipped replicate arrays were combined and normalized as log2 transformed ratios that were then averaged and subjected to centroid-linkage hierarchical clustering using Cluster 3.0 and displayed graphically to produce heat maps with Java Treeview.
7
miRNA Expression Profiling Using LNA Array
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An miRCURY LNA TM MicroRNA Array (version 11.0; Exiqon) was used. Total RNA (1 μg) was labeled with an miRCURY LNA TM MicroRNA Power Labeling Kit (Exiqon) according to the instruction manual, and then the fluorescently labeled RNA was dissolved in hybridization buffer (350 μL) from the kit. The solution was incubated at 95 ° C for 2 min, slowly cooled down to room temperature, and applied to the array slide. The slide was further incubated at 56 ° C for 16 h, and washed with the washing buffer from the kit as described in the manual. Finally, the washed slide was dried, and scanned with a GenePix 4000B (Axon Instruments). The obtained data were normalized and analyzed using an Array-Pro Analyzer (version 4.5; Media Cybernetics) as reported previously [19] .
8
Microarray Data Analysis Protocol
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The arrays were scanned using Axon GenePix 4000B scanner and the images of the array were analysed using GenePix Pro analysis software (version 5.0) (Axon Instruments, Foster City, CA) as indicated previously [32]. Briefly, subtract and offset method was used to correct the array background [36 (link)] and LOESS and scale-normalization methods were used to normalize differences within array variations [37 (link), 38 (link)] and between the arrays, respectively. A mean log2 transformed value of (Cy5/Cy3) was calculated from three replicates and the respective dye-swaps to obtain one value per target. Differentially expressed genes were identified using linear models for microarray data [39 ]. Genes with average log2 expression value > 0.65 and ≤ − 0.65 fold change and p < 0.05 and adjusted p value (FDR) < 0.2 were considered as differentially expressed genes.
9
Microarray Data Extraction and Analysis
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After recall, the microarray slide was scanned with GenePix 4000B (Axon Instruments, Forester City, CA) at 100% of laser power with 3 different PMTG settings from 800, 900, and 1,000 as detection sensitivity settings. Photobleaching was very minimal at the scanner settings. Signal intensities were measured with GenePix® Pro 6.0 microarray image analysis software (Axon Instruments, Forester City, CA) with 10 μm of pixel size as a detection sensitivity. The obtained data were saved as csv (comma delimited) format for data analysis. Background-subtracted intensity (BSI) values were used in the subsequent analyses. Statistical analyses were performed using statistical package R (version 2.8.0) [21 ].
10
Molecular Changes in Orthodontic PDL
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To investigate molecular changes in the PDL tissue, right- and left-side tissue blocks containing the only first molar roots were extracted 1 week after the application of orthodontic force. Total RNA was isolated using TRIzol Reagent (Molecular Research Center, Cincinnati, OH, USA) and was quantified using spectrophotometry. Profiling of gene expression was performed using Agilent 22K 60-mer oligonucleotide microarray slides (Digital Genomics, Seoul, Korea), which contained approximately 22,000 distinct sequences. The fluorescent-labeled complementary RNA (cRNA) was prepared by amplification of the total RNA in the presence of deoxyuridine triphosphate (dUTP), followed by the coupling of Cy3 (experiment) and Cy5 (control) dyes (Amersham-Pharmacia, Uppsala, Sweden). An Oligo Microarray Kit was hybridized with the fluorescent-labeled cRNA at 60℃ for 16 hours. The DNA chips were scanned using GenePix 4000B (Axon Instruments, Union City, CA, USA), and the scanned images were analyzed using GenePix Pro 3.0 software (Axon Instruments) to obtain the gene expression ratios. The logged gene expression ratios were normalized by LOWESS (locally weighted scatter plot smoother) regression.9 (link)
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