I tagged two proteins (A and B) of interest that i seek to study their interaction with Myc and HA respectively. From input, myc tagged protein was robustly present in my samples. Using myc magnetic beads, I was unable to IP protein A using anti-myc antibody. However, when I pulled down protein B using HA magnetic beads, I could observe protein A interacting with protein B (although the band was very faint).
I do not understand why the protein A that failed to IP could be interacting with protein B;
Hi Nwife,
a possible explanation for this effect could be, that in the native (IP)-condition the epitope of the myc-tag is sterically inhibited, in the denatured (WB)-condition the epitope is accessible for your antibody, and you "see" the myc-tag. In outher words, in the native condition the myc tag is burried in the protein "A". Sometimes it make sense to use a bigger tag like e.g. GST for pull-down or IP experiements or change the position from the c-terminal end to the n-terminal end (or the other way round).
Best,
Carsten
