Hi everyone, can you help me with this immunoprecipitation? Why also in the control there is the presence of Raptor? This control is made by no-adding the primary ab, isn't it? So the protein raptor not precipitate with the beads... And why we detected the IgG heavy chain if we use an ab against Raptor in the WB? Thanks to everyone will answer me!
Hi!
Take my reply with a pinch of salt, as I'm not 100% sure I'm correct. In the control, we do not use mTOR abs to check whether or not mTOR (or Raptor) will unspecifically bind to the precipitation beads. This result is telling us that some Raptor (or mTOR) has bonded to the beads aspecifically, but its enrichment is clearly visible when we do actually use mTOR abs in the "actual" experiment.
For the second question, since WBs usually use two antibodies (a primary antibody that is specific to some epitope on unfolded Raptor, plus a secondary antibody to detect the primary antibody), I'm guessing that the primary antibody used for this particular WB was from the same species as the one used for immunoprecipitation, so, using a single secondary antibody, you are both detecting the primary IgG bonded to Raptor plus the IgG that got washed away from the beads used to precipitate mTOR.
Please do correct me if I'm wrong, as that means I haven't understood something (or everything :D ) clearly.
Cheers!
Lane 1 is 10% input (total protein extract), lane 2 is IP with an Ab a-mTOR.
lane 1 --> Raptor is present in the protein extract
Lane 2 --> Raptor has been IP along with mTOR
Ig in lane 2: these are the Ig used for the IP of mTOR
Since Ig signal is quite high, Luca's explanation makes sense. Even if the 1° Ab used for the IP and the WB would be from different species, very high quantity of proteins give rise to non specific binding.
I hope this explanation will help!