I have a cell surface protein showing differential expression based on immunofluorescence and flow cytometry. Does anybody have any suggestion why it is so?
Thanks.
The expression certainly does not change depending on the technique alone.
The difference you are observing is likely due to differences in sensitivity and specificity of your detection method.
I agree with the first reply.
FACS should be a more quantitative method for measuring surface expression. Quantifying data in IF is often more tricky and prone to errors.
Staining in FACS is also often performed on non-fixed cells. Thus, one a has to be careful with the temperature and conditions of the experiment. If performed at room temperature there is chance that receptors could be internalized before antibody addition or that the assay be perturbed in many other ways downstream. Usually staining of surface proteins for FACS will be performed on ice.
The first two answers are very valid. It's all down to sensitivity of the methods.
I agree with previous answers. Compared to immunofluorescence the flow cytometry is a more sensitive method and is typically performed with non-fixed cells. If you have used the same antibody to detect your antigen of interest with both methods, the fixation method used for preparation of a specimen for immunofluorescence analysis could be inappropriate/suboptimal for a sensitive and specific detection of the antigen. In which method was the expression of your antigen higher or more specific? Please, provide some more details about the procedures used in order to get more specific answer.
Expression does not depend on the detection method. Flow cytometry used to be more sensitive than IFA, specially if any epitope suffer damage during cell fixation.
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