|
| Status |
Public on May 08, 2014 |
| Title |
BRAA D1B |
| Sample type |
other |
| |
|
| Source name |
BRAA from B-Cells isolated from Mouse Spleen Cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: MRL/lpr
cell type: B cell
antigenic target: Autoreactive to brain antigen
|
| Treatment protocol |
Not applicable
|
| Growth protocol |
Brain-reactive autoantibodies were created using hybridoma technology. The B-cells from an autoimmune mouse and myeloma cells were fused and cultured.
|
| Extracted molecule |
other |
| Extraction protocol |
The antibodies produced in the supernant from these hybridomas were tested for reactivity to brain antigen using ELISA and those antibodies that displayed reactivity above 0.1 OD were retained for testing on microarray.
|
| Label |
Alexafluor 647
|
| Label protocol |
Brain-reactive autoantibodies bound to the arrays were detected using goat anti-mouse IgG biotinylated secondary antibody followed by streptavidin conjugated with AlexaFluor 647.
|
| |
|
| Hybridization protocol |
Each slide was blocked with buffer containing mercaptohexanol for one hour followed by a one hour incubation with each monoclonal antibody. The slides were washed with TBST and then incubated with 6.67μM of goat anti-mouse IgG biotinylated secondary antibody at 37oC for one hour. 5nM of streptavidin conjugated with AlexaFluor 647 was added to the slides, the slides were washed and then spun dry before scanning.
|
| Scan protocol |
A Perkin Elmer Scan Array laser Scanner was used to scanned the slides (543nm emission, 565 nm absorption, 75% PMT, 100% laser power).
|
| Description |
Goat anti-mouse IgG biotinylated secondary antibody was added followed by streptavidin conjugated with AlexaFluor 647.
|
| Data processing |
GenePix Pro 6.0 was used to produce a gpr file that was analyzed in GeneSpring 7.3.1.
|
| |
|
| Submission date |
May 07, 2014 |
| Last update date |
May 08, 2014 |
| Contact name |
Phillip Stafford |
| E-mail(s) |
phillip.stafford@asu.edu
|
| Organization name |
Biodesign Institute
|
| Department |
Center for Innovations in Medicine
|
| Lab |
Biostatistics
|
| Street address |
1001 S. McAllister
|
| City |
Phoenix |
| State/province |
AZ |
| ZIP/Postal code |
85287 |
| Country |
USA |
| |
|
| Platform ID |
GPL17600 |
| Series (1) |
| GSE57389 |
Diagnosis and early detection of CNS-SLE in MRL/lpr mice using peptide microarrays (GPL17600) |
|
