For the gel, it might be really hard to see the peptide as small things tend to blur terribly--better to look for shifts in the protein band. If I were you, I would do serial dilutions of the peptide at some constant, very visible protein concentration. Also you have to be sure that the protein/peptide charges work--and you can run basic (positive) proteins on native gel by simply switching the electrodes. Pay attention to what you put in your loading buffer as well, as this may alter binding.
JPK On Fri, Sep 14, 2012 at 8:46 AM, anita p <crystals...@gmail.com> 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 > -- ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu *******************************************
