I am working with antibody microarrays printed on epoxy glass. The slides are incubated with serum followed by a secondary antibody conjugated with a fluorescent dye. 10-15% of the time we are seeing a global decrease in fluorescent signal across an entire reaction well. We cannot determine the source of this signal inhibition.
Our serum is diluted (1:80) in 200mM NaPO4, 10% glycerol, 5% BSA, and 0.01% Tween-20 at pH 7.4. Our secondary antibody is diluted (1:2000) in 5% BSA, 1X PBS, and 0.05% Tween-20.
Could the glycerol or sodium phosphate be the root of these signal inhibitions? The signal inhibition only occurs in the presence of serum.
Daer Jonas!
Please You look at this article:
Article Fabrication of Homogeneous High-Density Antibody Microarrays...
Any viscous compound such as glycerol or high concentrations of BSA slows down the binding reaction. I would leave out the glycerol and reduce the BSA to 0.1% if you incubate these solutions and do not print them. Sometimes such variations are also caused by a different temperature or poorly mixed secondary antibody solutions.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies