This may be tricky if binding is too weak, but could you use some
ultrafiltration with, say, 5kDa filters? The idea is that peptide will
not go through if it is associated with protein. Another option is size
exclusion, although that is not always straightforward as weakly bound
ligands may come off upon dilution due to peak broadening. You are
using tris-tricine buffers, right?
On 09/14/2012 09:46 AM, anita p wrote:
Hi All,
I wanted some advice regarding mapping out Protein-peptide
interaction. The peptide is a 12 mer and the protein is 15kDa.
Invivo studies suggest that the peptide is binds the protein and helps
in transport.
Hence I feel it would perhaps transient binding.
I know that I should do ITC or BIAcore to show binding, but before
going to those techniques, I feel, running a native gel would perhaps
help. So the native gel can have lane1: protein, lane 2: peptide, lane
3: protein+peptide.
If the protein binds to the peptide then I should not see a band
corresponding to the peptide in lane 3.
But before I start this experiment, I wonder if any body has run 12
mer peptide on native gel, How long should I run... How much
quantity of peptide I should for the gel.
I wont be able to do western or pull-down, Equipment for native gel is
available to me.
kindly advice,
regards
Anita
