ProtoArray microarrays (v4.0; Invitrogen) were purchased and used according to the manufacturer’s instructions. Briefly, after blocking for 1 h at 4° C and washing, arrays were incubated in Quadriperm dishes (Greiner Bio One) placed on a horizontal shaker (50 rpm) for 90 min at 4° C with individual sera diluted 1:500 in 5 ml washing buffer (0.1% Tween 20 [vol/vol], 1% BSA [wt/vol] in PBS). After washing, binding of IgG was detected by incubation with Alexa Fluor 647 goat anti-human IgG (Invitrogen) diluted 1:2,000 in assay buffer for 90 min at 4° C. Arrays were washed again and dried by centrifugation. Arrays were scanned at 10-μm resolution using a microarray scanner (Axon 4200AL with GenePix Pro Software; Molecular Devices), and fluorescence was detected according to the manufacturer’s instructions. Images were saved as 16-bit TIFF files and analysis was performed using GenePix. The median net intensity in relative fluorescence units (rfu) was reported for each spot. Out of 9,481 antigens, 77 cancer-testis antigens were selected and analyzed for pre- and post-vaccine expression in the sera (Table 4 ).
Axon 4200al
Manufactured by Molecular Devices
Sourced in United States
The Axon 4200AL is a high-performance electrophysiology amplifier designed for reliable data acquisition. It features low-noise, wide-bandwidth amplification for accurate recording of electrophysiological signals.
Automatically generated - may contain errors
Lab products found in correlation
Quadriperm dishes, by Greiner (3 mentions)
Genepix pro software, by Molecular Devices (3 mentions)
Alexa fluor 647 goat anti human igg, by Thermo Fisher Scientific (1 mentions)
Protoarray human protein microarrays v5, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 conjugated goat anti human igg, by Thermo Fisher Scientific (1 mentions)
3 protocols using axon 4200al
1
Profiling Cancer-Testis Antigens via ProtoArray
2
Protein Microarray Profiling of Sera
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ProtoArray Human Protein Microarrays v5.0 (Invitrogen, Carlsbad, CA, USA) were purchased and used according to the manufacturer’s instructions.
After blocking for 1 h at 4 °C and washing, arrays were incubated in quadriPERM dishes (Greiner Bio-One, Frickenhausen, Germany) placed on a horizontal shaker (50 rpm) for 90 min at 4 °C with individual sera diluted at 1:500 in 5 mL washing buffer (0.1% Tween 20 [v/v], 1% BSA [w/v] in PBS). After washing, binding of IgG was detected by incubation with Alexa Fluor 647–conjugated goat anti–human IgG (Invitrogen, Waltham, MA, USA) diluted 1:2000 in assay buffer for 90 min at 4 °C. The arrays were washed again and dried by centrifugation. Arrays were scanned at a 10-μm resolution on a microarray scanner (Axon 4200AL with GenePix Pro Software; Molecular Devices, Sunnyvale, CA, USA), and fluorescence was detected according to the manufacturer’s instructions. Images were saved as 16-bit TIFF files, and analysis was performed using GenePix. The median net intensity in relative fluorescence units is reported for each spot.
After blocking for 1 h at 4 °C and washing, arrays were incubated in quadriPERM dishes (Greiner Bio-One, Frickenhausen, Germany) placed on a horizontal shaker (50 rpm) for 90 min at 4 °C with individual sera diluted at 1:500 in 5 mL washing buffer (0.1% Tween 20 [v/v], 1% BSA [w/v] in PBS). After washing, binding of IgG was detected by incubation with Alexa Fluor 647–conjugated goat anti–human IgG (Invitrogen, Waltham, MA, USA) diluted 1:2000 in assay buffer for 90 min at 4 °C. The arrays were washed again and dried by centrifugation. Arrays were scanned at a 10-μm resolution on a microarray scanner (Axon 4200AL with GenePix Pro Software; Molecular Devices, Sunnyvale, CA, USA), and fluorescence was detected according to the manufacturer’s instructions. Images were saved as 16-bit TIFF files, and analysis was performed using GenePix. The median net intensity in relative fluorescence units is reported for each spot.
3
Identifying Autoantibody Biomarkers for ACR
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To identify significant autoantibodies, present at different concentrations in patients with ACR after transplantation, we performed the microarray analysis using serum samples from HCV-positive transplant recipients with ACR and control groups. ProtoArrays microarrays (v4.0; Invitrogen) were used to identify candidate autoantibodies to predict ACR according to the manufacturer's instructions. Respective sera were diluted 1:500 in washing buffer (0.1% Tween 20, 1% bovine serum albumin in phosphate buffered saline (PBS)). After blocking for an hour, the arrays were incubated with diluted sera for 90 minutes at 4°C in Quadriperm dishes (Greiner Bio One) using a horizontal shaker (50 rpm). After washing, the arrays were incubated with 1:2000 diluted Alexa Fluor 647 goat antihuman IgG for 90 minutes at 4°C to detect binding of IgG. The arrays were scanned at 10-μm resolution using a microarray scanner (Axon 4200AL with GenePix Pro Software; Molecular Devices). Fluorescent images were saved as 16-bit tif files and analyzed by GenePix. The median intensity of each spot in relative fluorescence units was recorded.
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