I want to check the CD19 marker on the surface of the mouse spleen B cell by flow cytometry. What secondary antibody can I choose that does not have non-specific connections?
Very first thing, why you want to purchase an antibody without fluorescence tag. Go for a fluorescence tag antibody so that you need not to do indirect staining and 1 extra step.
If you have an unconjugated antibody with you then you need to buy the secondary antibody compatible to primary. Usually on the technical data sheet, you will find the compatible suggestions.
To check the nonspecific binding of secondary antibody you should run experiment;
1 tube of unstained, 1 tube for secondary only (to check non specific binding) and last 1 for primary antibody staining followed by secondary.
Hope this is usefull.
Very first thing, why you want to purchase an antibody without fluorescence tag. Go for a fluorescence tag antibody so that you need not to do indirect staining and 1 extra step.
If you have an unconjugated antibody with you then you need to buy the secondary antibody compatible to primary. Usually on the technical data sheet, you will find the compatible suggestions.
To check the nonspecific binding of secondary antibody you should run experiment;
1 tube of unstained, 1 tube for secondary only (to check non specific binding) and last 1 for primary antibody staining followed by secondary.
Hope this is usefull.
There are many nice CD19 conjugated antibodies available, I would prefer to use that to avoid 2 step staining or staining with 2nd antibody. Antibodies from Biolegend are comparatively cheap and works really great. In case if you must add 2nd antibody other factors such as type of flow machine, other markers in antibody cocktail etc. will determine which antibody (fluorophore) you should use for staining.
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