I recently used a biotin-streptavidin antibody (conjugated in house) as well as a preconjugated AlexaFluor 647 antibody, upon analysis it has proven to be difficult to set a threshold without a no primary control.
First of all, you need to show deficiency of non-specific binding of your antibody, so select the cell types wish is deficient of the marker.
And then proceed with your cell of interest for staining.
Not every time you need to run the negative control. Once you show the negativity of your antibody on deficient cell types. And rest of the time this negative control may explain the story.
Do you have a cell type or tissue that is known to lack the antigen which could be used as the negative control?
First of all, you need to show deficiency of non-specific binding of your antibody, so select the cell types wish is deficient of the marker.
And then proceed with your cell of interest for staining.
Not every time you need to run the negative control. Once you show the negativity of your antibody on deficient cell types. And rest of the time this negative control may explain the story.
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