So what they did is they extract the proteins from the nerve terminal. So in fact, they did not use all the proteins. They only extract the protein from the so-called synaptosome, where that is the protein that enrich in the synapse. And that will be good because you postulate, you work on the synapse to inhibit transmitter release. Rather than using western blot, so western blot has a specific meaning. Western blot meaning that you use the antibodies to detect which proteins. They do not use western blot because they don't know which protein. Rather than they are relying on the fact this is a putative protease. The protease what it's doing is to cut things into smaller band. So what they did is they used an SDS-PAGE that they can extract a protein, treat it with the toxins, and then look at what has been changed in the molecular weight. And then when you are running the SDS-PAGE, all the protein are denatured, so they will shift that molecular weight. And it looks so many bands but let's just looking at two of them. You're just looking at, for example, this one, this toxin plus EDTA and look at this band here. And then you'll look at the adjacent one, is that only the toxin, the light chain, which is effective ones, was using to treat this extract. So you look at this one and these two. And then so why are they adding a toxin, adding EDTA? Because the EDTA can chelate zinc. EDTA can chelate divalent ions, magnesium, calcium, zinc. And after chelating the zinc, which is a essential catalytic cofactor, presumably they will completely eliminate the protease activity. So just compare the band here and then this band without the EDTA but with the light chain toxin. Just look at these two band. And then tell me what do you think the difference between these two bands. Did you identify the putative substrate? What are you going to do? Let me just say, this band here is showing this protease light chain alone that has zinc that it will cleave something and this one is a control showing that with the EDTA to eliminate this protease cut activity. In the protease inhibit condition, that's one band here. Right? And this band is eliminated, it's gone in the protease treatment condition. Instead there's two additional bands coming up. Then it's likely that this band get cleaved by this into these two fragments. So considering molecular weight. So what is the molecular weight here? The molecular weight probably is like ten. And then this one is less than eight. So probably this is six or seven. So 17 KB and this in between 14 and 20, maybe it's 16, so actually it makes sense, right? So then if this is true, you postulate that this band here might be the substrate for these proteins. And then it will get cleaved into two fragments under that condition. So then what? So proposed to use this biochemical approach, and then they indeed follow his proposal, do that, and then identify the putative molecular substrate. Now what are you going to do? So now you identified a putative bend here, it might be the substrate. Now to the problem is or the big elephant in the room is, what exact protein it might be. You're only seeing a band there, you don't know the identity. You know the roughly molecular weight. You don't know that sequence, what protein, what gene encoding this protein, right? This is a problem. So the urgent thing is to identify which molecule it is, which protein it is, right? So proposes using a mass spectrum analysis to identify the protein. That's cool. Although in 20 years ago, or more than 20 years ago, they don't have that kind of resolution. There's no such high resolution MSMS to helping you to identify the sequence. But instead, they can just prove a lot of proteins, and then using this old-fashioned end terminal Edman degradation method to sequence the protein. But the purpose is the same, to identify which protein it is. But there's even more problems. Are you going to only sequence this one? Where does the cleavage happen? Where does it happen? At which site because you propose is this putative protease, that it will cut this band into two, right? Then this is just a molecular weight size as point out. It may make sense, but again, you are lacking of the bulletproof evidence. So since you can sequence this gene, then why don't you just do a little bit more work. You sequence this too, as well. Today if you also sequence this too, what is your prediction? So the prediction will be that if this one gets cleaved into two parts, right? So once you identify this protein by sequencing, you will find out these two are part of the same sequence of this one. And again, in the end terminal Edman degradation method, in this way Edman degradation method is a powerful method, because it will allow you to directly sequence the end terminal of this peptide. So essentially, by sequence these two, you can identify which site are the cleavage site? The mass spectrum analysis alone actually is difficult to help you to identify the cleavage site, because what mass spectrum analysis is doing is just to randomly sequence part of the peptides. It doesn't tell you the exact end terminal. So this method can help you doing that. So that's what they did. So again, they sequence this peptide and the small fragment and they found. Well, it turns out that it's the synapto that was in this site. And then they also sequence these two, they identified a putative cleavage site. Again, consistent with this being cleaved into these two fragments. These two fragments are part of this synaptic or you call it one one, one two. It's the same molecule, have a different names. Then what? You sequence it. You use the mass spectrum or end terminal degradation. You identify it and then you identify these two fragment also consistent is part of it and then you identify the cleavage site. And then what are you going to do? Great. So first, you demonstrate that this is the putatively this substrate and then it gets cleaved into two fragment. You identified the cleavage site. But with this experiment, do you have the direct evidence it is this toxin that cleaved synaptic? There's an alternative possibility. Maybe this toxin cleaved some other protease, activated some other components in this extract. And this other component may be another protease that it will cleave this into synaptic [INAUDIBLE] and that get into these two fragment. In apoptosis field, the cut space that it is working, actually people know that the cut space will activate a downstream cut space. And then the downstream cut space will fully activate to cleave other components. Only this evidence did not prove to you that it's this toxin directly cleaved that into the two parts. Maybe there are some additional components. So how are you going to do it?
