tissue: Sera molecular: Autoantibody age (years): 60 Sex: Male al: 27.49
Extracted molecule
protein
Extraction protocol
1 ml venous blood per subject was collected into a promoting coagulant vacuum blood tube. The top layer serum samples after centrifugation at 3000 rpm for 10 min at 4 °C within 4 h after collection were sucked out and stored at −80 °C until use.
Label
Cy5
Label protocol
1:2,000 diluted Cy3-conjugated goat anti-human IgG (Jackson, 109-165-008) and Alexa Fluor 647-affinipure donkey anti-human IgM (Jackson, 709-605-073) to fluorescently label IgG and IgM
Hybridization protocol
After blocking 1h, HuProt array was incubated in 3 ml 1:1,000 diluted serum sample to capture corresponding autoantibodies to each protein spot, and followed with 1:2,000 diluted Cy3-conjugated goat anti-human IgG (Jackson, 109-165-008) and Alexa Fluor 647-affinipure donkey anti-human IgM (Jackson, 709-605-073) to fluorescently label IgG and IgM of captured autoantibodies, respectively.
Scan protocol
Scanned using the GenePix 4000B Microarray Scanner (Molecular Devices, Sunnyvale, CA, USA) and analyzed with GenePix Pro 6.0 software (Molecular Devices, Sunnyvale, CA, USA)
Description
IgM
Data processing
The signal intensity of each protein spot was calculated as the ratio of foreground to background signal intensity. Each protein was duplicated in pairs, thus, the signal intensity of each autoantibody was calculated as the mean of both protein spots.
