Okay so am I correct in thinking that in the first row, you have pluses across because anti-flag antibody was present because it had nothing to bind to?
I apologize - I meant first column. I'm sorry for any misunderstanding that caused.
So with the experimental design, the graph becomes a lot easier to explain. So you have four different conditions here, namely MHC (full peptide), S1, S2, and the isolated motor fragment (HMM). So let's go through these one-by-one. So MHC is the full peptide that the scientists synthesize. It's initially unfolded and they incubate it with these chaperones. Now, apparently these chaperones aren't just normal chaperones - they have been engineered to have a radioactive tag (the FLAG-anti-FLAG conjugate pair). So then they run everything through a gel. Their visualization technique is radiation-based, so the only bands that will appear will correspond to either free chaperones (with the anti-FLAG tag) or chaperones with the peptide bound inside. Nothing else is radioactive. Since we don't see the former, it's possible that after the peptide bound the chaperone, they washed everything with non-tagged chaperones so that only the peptide-bound chaperones will show on the gel. In any case, that's how they visualize whether the peptide is bound.
So let's focus on MHC now. MHC is the synthetic full, unfolded peptide. So it contains a motor domain within itself and will need the chaperone to fold. In the first column, they incubate with just anti-FLAG antibody. Since the chaperones here have not been modified with FLAG, the anti-FLAG obviously won't bind to anything and will just get washed out before they load the sample onto the gel. Since anti-FLAG doesn't bind to anything, then you wouldn't be able to see anything on the gel because there's no radioactivity. So this is just a negative control to make sure that you don't have non-specific anti-FLAG binding. In the second column, they treat with radioactive-labeled Unc45b (from the passage, I'm deducing that it's a Unc45b that already has the anti-FLAG tag on it). The MHC peptide binds as you would expect it to and you see it on the gel. Similarly, the Unc45b/Hsp 90 chaperone also binds to MHC.
Now let's move to S1 and S2. Recall that S1 contains the motor domain and S2 does not. Going through the same steps, you can see that both chaperone complexes bind to S1 but not S2. So this suggests that the presence of the motor domain is necessary for chaperone binding. But one should be careful here because it's not exactly clear whether S1 and S2 differ
only by the presence and absence of the motor domain or if there are more sequence differences. If there are more sequence differences, then one could only say that
something on S1 is necessary for chaperone binding. Anyway, this is the crux of the experiment.
Finally, HMM is kind of a negative control as well, to test whether your tagged chaperones are binding non-specifically. That is, are they binding only to proteins that need to be folded or do they bind to folded proteins containing a certain sequence as well? This is a test of tertiary structure. That is, MHC and HMM presumably have the same primary sequence. Therefore, does the
3D structure that primary sequence is in matter or not? Here, we see that the chaperones do
not bind to HMM. This means that the 3D structure of the primary sequence does matter because the chaperone is binding to some sequence in unfolded MHC and while that same primary sequence is in HMM, it's not binding to the folded form. So the chaperone is only binding to the unfolded form, which is what you want.
