Protein lysates were prepared by the BCM Antibody-Based Proteomics Core for reverse phase protein array assays. The Aushon 2470 Arrayer (Aushon BioSystems) with a 40 pin (185 μm) configuration was used to spot lysates onto nitrocellulose-coated slides (Grace Bio-Labs). The slides were probed with 220 antibodies against total and phospho-proteins using an automated slide stainer (Dako). Primary antibody binding was detected using a biotinylated secondary antibody followed by streptavidin-conjugated IRDye 680 fluorophore (LI-COR). Fluorescent-labeled slides were scanned on a GenePix AL4200 and the images were analyzed with GenePix Pro 7.0 (Molecular Devices). Background-subtracted total fluorescence intensities of each spot were normalized for variation in total protein (Sypro Ruby) and nonspecific labeling. For adipokine analysis, tissue lysates from four samples were pooled (125 μg/sample) and incubated with membranes from the Proteome Profiler Mouse Adipokine Array Kit (R&D Systems #ARY013) according to the manufacturer’s instructions.
Irdye 680 fluorophore
Manufactured by LI COR
The IRDye 680 fluorophore is a near-infrared dye used for various labeling and detection applications. It has an absorption maximum at 680 nm and an emission maximum at 700 nm, making it suitable for detection in the near-infrared region of the electromagnetic spectrum.
Automatically generated - may contain errors
Lab products found in correlation
Automated slide stainer, by Agilent Technologies (3 mentions)
Genepix al4200, by Molecular Devices (3 mentions)
Genepix pro 7, by Molecular Devices (3 mentions)
Nitrocellulose coated slides, by Grace Bio-Labs (3 mentions)
Proteome profiler mouse adipokine array kit, by R&D Systems (1 mentions)
Tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
3 protocols using irdye 680 fluorophore
1
Reverse Phase Protein Array Analysis of Adipokines
2
Reverse Phase Protein Array Analysis
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Protein lysates were prepared by the BCM Antibody-Based Proteomics Core for reverse phase protein array assays. The Aushon 2470 Arrayer (Aushon BioSystems) with a 40 pin (185 μm) configuration was used to spot lysates onto nitrocellulose-coated slides (Grace Bio-Labs). The slides were probed with >220 antibodies against total and phospho-proteins using an automated slide stainer (Dako). Primary antibody binding was detected using a biotinylated secondary antibody followed by streptavidin-conjugated IRDye 680 fluorophore (LI-COR). Fluorescent-labeled slides were scanned on a GenePix AL4200 and the images were analyzed with GenePix Pro 7.0 (Molecular Devices). Background-subtracted total fluorescence intensities of each spot were normalized for variation in total protein (Sypro Ruby) and nonspecific labeling.
3
Multiplexed Protein Profiling of Cell Lysates
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Protein lysates were prepared with Tissue Protein Extraction Reagent (Thermo Fisher) containing protease and phosphatase inhibitors (Roche). The Aushon 2470 Arrayer (Aushon BioSystems) with a 40 pin (185 μm) configuration was used to spot lysates onto nitrocellulose-coated slides (Grace Bio-Labs). The slides were probed with 220 antibodies against total and phosphoprotein proteins using an automated slide stainer (Dako). Primary antibody binding was detected using a biotinylated secondary antibody followed by streptavidin-conjugated IRDye 680 fluorophore (LI-COR Biosciences). Fluorescent-labeled slides were scanned on a GenePix AL4200, and the images were analyzed with GenePix Pro 7.0 (Molecular Devices). Total fluorescence intensities of each spot were normalized for variation in total protein (Sypro Ruby) and nonspecific labeling.
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