Prey protein band bleeds, IgG band normal? - (Aug/28/2013 )
Very frequently, when I run my Co-IP samples, the prey protein band will bleed into adjacent lanes. Conversely, the input for these proteins retains good lane discipline and the bands are clearly separated.
Moreover, the protein I pull down does not experience this issue, and the heavy/light chain bands also resolve quite nicely into separate lanes. As a corollary, the pulled-down protein has a similar MW to the prey proteins (e.g., within ~15kD): if there was a problem with the gel, then the IgG should look funny too, and most definitely the pulled-down protein.
Thanks for any help you can provide.
the only things i can think of are:
too much protein loaded
additives in the co-ip buffer (eg salts, detergents)
pH of the co-ip buffer (its effect on the sds sample buffer)
Thanks for the ideas. My guesses are between too much protein loaded and pH of the co-IP buffer. Specifically, I am worried that the beads may affect the pH. Very frequently, after I boil my samples in sds sample buffer, the liquid in IP/beads tubes change color from blue to green (and, in extreme cases, yellow). However, the lysate/input tubes stay blue. Next time I do IP I will use a newer aliquot of Tris (my Flag lysis buffer calls for 50mM so I probably can't use any more).
if the tracking dye is turning green to yellow then the pH is too low. you can add a few ul (or less) of 0.5M (or lower) tris base (or pH 9) to the sample to raise the pH until the dye turns blue.
the lower pH does have an effect on migration.