I recently got a problem with my secondary Ab, it is PE goat anti-rat IgG and it stained B6 mouse liver lymphocytes non-specifically. But when I incubated primary Ab and secondary Ab for 3 hours and then directly stain cells, this disappears. I was wondering if it is ok to do like this?
You may do that... But make sure you are using low concentration of primary antibody.....
Again it varies from antibody to antibody.... It may reduce the affinity of primary antibody binding....
It may be possible that, the incubation of primary and secondary antibodies together in a solution causes precipitation and loss of both antibodies from the solution, which results in no binding of antibodies to the cells. To deal with non-specific binding, either use isotype control or blocking with serum that matches (goat serum for secondary antibody from goat) the secondary antibody. Goodluck
Hi Zhengzuo,
a) I wonder if you blocked the FC receptors on the lymphocytes prior to addition of ABs. The FC regions of the ABs can react to FC receptors on lymphocytes, which may explain the none-specific binding of your secondary.
b) It is better to have fluorochrome conjugated ab for flow. If it's not available for your protein, use conjugation kits to link up your primary and secondary for better staining consistency.
c) In case you haven't thought about it, including a cell type as positive control for the protein might help you trouble-shoot this problem faster.
All the best
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining