Hi,
An antibody specific to the extracellular domain of HER2 receptor was produced. It is used as both target protein and primary antibody on SKBR3 cell ELISA assay in order to confirm its binding activity.
We assume that antibodies that bind to the receptors are internalized, along with their receptors by endocytosis after cell fixation, and it can be one of the reasons of unsatisfactory ELISA results.
What happens to the biological phenomena of cell and its cell surface receptors like epidermal growth factor receptor (EGFR) family when fixed with paraformaldehyde? Does internalization of HER2 receptor/antibody complex occur after fixation?
Any help will be appreciated.
Paraformaldehyde can sometimes lead to permeabilization of the plasma membrane. Both the elapsed time after adding the PF, the pH of the PF solution affects fixation parameters, especially the balance between tissue penetration and cross-linking. Depending on the protocol, one possibility is that antibodies may bind to receptors in endocytic vesicles.
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