I have been using 3 different primary antibodies (2 anti-TLR3 and 1 anti-Flag) to identify TLR3 in 293T cells, after transfecting the cells with TLR3-Flag plasmid.
The problem is that I have a high molecuar weight band in the region expected for TLR3 expression.
My questions are:
Does anybody have a similar experience?
I am wondering if the antibodies are picking up any other TLR. Are this cells TLR negative?
We studied the TLR3 molecule in transfection on HEK293 or 293T to develop antibodies against this molecule. None of the 30 different hybridoma clones that we do recognize the HEK293 cell.
We also found no TLR3 gene by PCR on this cell.
It is possible that the band you are observing is an artifact associated with the antibody you are using
TLR3 is often present on tumor cells but very little on the cell lines used in transfection.
Thank you for your reply Jean-Jacques. Do you know if HEK293T cells express any other TLRs?
We have produced other TLR monoclonal antibody libraries from transfected HEK293 T cells. None of these antibodies bind the cell 293.
This concerns TLR2,4,7,8,9 and 10.
I do not have information for other TLRs that are often less studied.
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