non-specific binding? - (Feb/09/2006 )

I'm having trouble with a rabbit antiserum against a peptide derived from the cytoplasmic tail of a membrane protein. The antibody appears to be labeling cells transfected with the protein even though the cells have not been specifically fixed/ permeablized prior to antibody labeling. It doesn't label untransfected cells where the protein is not present. I've tried changing the secondary antibody that we use for detection, but that didn't make a difference. This occurs with labeling for both flow cytometry and immunofluorescence microscopy.

We made this antiserum to use as a negative control for surface expression since it shouldn't label the protein unless it can enter the cells.

Specifically, we are using cos-7 lipofectamine transfected cells. For flow cytometry, we release the cells from the plates with trypsin and use PBS with FBS and sodium azide as our labeling and wash buffer. For IFA, we label the live cells with the same buffer. We label for 1 hour at 4 degrees for each antibody.

It's been suggested that maybe the protein is upside down, but my usual antibodies against the extracellular domain also label. It was then suggested that the protein has multiple forms and can feed through the membrane multiple times, but transmembrane prediction models don't predict this as possible. I think that there is something screwy with the cells, antiserum or method that I'm using.

Any thought? suggestions?

Thanks in advance for any advice. This is making me crazy

I'm having trouble with a rabbit antiserum against a peptide derived from the cytoplasmic tail of a membrane protein. The antibody appears to be labeling cells transfected with the protein even though the cells have not been specifically fixed/ permeablized prior to antibody labeling. It doesn't label untransfected cells where the protein is not present. I've tried changing the secondary antibody that we use for detection, but that didn't make a difference. This occurs with labeling for both flow cytometry and immunofluorescence microscopy.

We made this antiserum to use as a negative control for surface expression since it shouldn't label the protein unless it can enter the cells.

Specifically, we are using cos-7 lipofectamine transfected cells. For flow cytometry, we release the cells from the plates with trypsin and use PBS with FBS and sodium azide as our labeling and wash buffer. For IFA, we label the live cells with the same buffer. We label for 1 hour at 4 degrees for each antibody.

It's been suggested that maybe the protein is upside down, but my usual antibodies against the extracellular domain also label. It was then suggested that the protein has multiple forms and can feed through the membrane multiple times, but transmembrane prediction models don't predict this as possible. I think that there is something screwy with the cells, antiserum or method that I'm using.

Any thought? suggestions?

Thanks in advance for any advice. This is making me crazy