I worked very hard this summer, trying to gain significant progress on my aims.  This was particularly important, because what I was working on, if it worked, would provide some key in vivo insights for a paper that we’re trying to publish on this protein-protein interaction.  Luckily, several experiments did work out! (or at least as well as these things tend to work )  It’s nice to close out this particular chapter of research with that note, although I will be going back to do some more investigating in the fall, of course.

We also closed out this summer with a joint lab meeting between the Kerscher Lab, a SUMO lab at Johns Hopkins, and a SUMO lab at the NIH.  Four of us packed into a car and headed up to the NIH for the day, where several members of each lab gave presentations of their work.  There was so much science jam-packed into 5 hours in that one little conference room.  It was really neat to hear the many different aspects of different people’s work on this one system (the other two labs do most of their work in other mammalian systems or human cell lines).  I also had the opportunity to present the summary of our research on this novel interaction we’ve identified and characterized, including pieces of my own work on it.  I enjoyed putting together the presentation, summing up all of our research in a logical fashion to tell the story of our science.  I felt honored to be able to present that story, which contains key work from several people who have been in and out of the lab.  This project began as the work of a previous Master’s student in the lab, was spurred on by a tiny piece of data gathered from Dr Kerscher’s genetic analysis lab, had large contributions from two previous undergraduates in the lab, and now has some from me, our current grad student, and our postdoc.  Though this might be more unusual in a larger university/government lab, it’s neat to think that so many people’s work went into this project.  Collaborative science!

I’ve spent some time lately reading other related articles, trying to get a sense of what people are discovering now, how it might relate to our work, and how that might inspire us to investigate something else or look at something in a new light.  Looking back at the notes I’ve taken, they’re full of crazy abbreviations of proteins and complexes and pathways, but for the most part, I understand it all.  It makes me feel privy to a secret world of research science.  One particular thing that’s clicked for me recently is how powerful yeast genetics can actually be.  Manipulating or deleting a gene of interest might have observable effects on the growth phenotype of the yeast, giving you insights into the gene’s importance.   For example, yeast cells without SLX5 grow extremely slowly and are far more sensitive to DNA damaging stresses. But beyond that, you can learn about protein-protein interactions if you compare the growth phenotypes of yeast with no deletions, with one gene deleted, and with both genes deleted.  For example, an SLX5 deletion is unhealthy, but if you also delete the gene of one of its interactors, it may be “rescued” and be healthy again.  The theory behind that logics that getting rid of one protein might be bad because then it can’t regulate/work with the other protein that it’s interacting with, so that misregulated protein wreaks havoc.  But if you get rid of the other protein too, than you might be a little better off, because you don’t have the misregulated protein mucking things up because it’s not there at all.  Kind of confusing, and I can see how it might not always be true for all interactions, but nonetheless, since yeast are so easy to genetically manipulate, looking at those genetic interactions can be an extremely powerful tool.  Numerous novel, important proteins have been discovered in screens similar to the one I described.  Like I said earlier, understanding them makes me feel like I understand this secret, obscure world that occupies the pages of scientific journals, which is so cool.

So with all that kicking around my brain, I want to be more aware of trying to use those genetic approaches in my coming research.  I want to gather what I’ve learned from other papers, plus what we’ve found, and extrapolate a larger picture for it – the creation of transient protein complexes using our post-translational modifiers in response to DNA damage, or something like that.  I want to finish up a couple of things on this project, and start working on a new one, tweaking some techniques and getting creative.  Regardless of how everything goes, I know that I’ve learned so much about research science and how to do it, and I will take that knowledge and appreciation of research with me where ever I go in life, whether I go into a hard-core research career or not.

Now that I’ve left lab for the summer, here are the final updates on some of my aims:

Protein Extraction and Pull-down

After a lot more buffer tinkering, an amusing change of our epitope tag + affinity bead combination, and some new strains, I think I’ve finally got it!  Or at least the most promising one yet, which is extremely encouraging and at least relatively satisfying to leave behind for these next couple weeks.  Some more detail about the changes that I made:

• I started using a more specific elution buffer.  My proteins of interest are supposed to be bound to the beads based on their tag or if they’re interacting with the protein with the tag.  However, some proteins bind non-specifically to the  beads (due to their interactions with the sugar that the bead base is made up of, or other non-specific way).  My old method of getting proteins off after my washes was by adding LDS-sample buffer and boiling the beads.  The sample buffer, a detergent with both a charged and uncharged section on it, denatures the protein’s native structure, causing anything bound to the beads to come off of them.  The problem is that it causes everything bound to come off – including those proteins that are non-specifically bound, thus contaminating our sample.  To circumvent this issue, since I’d been getting contaminants in the control lanes, I started using high levels of imidazole in my elution buffer when using TALON affinity resin.  TALON affinity resin contains immobilized cobalt ions, which is what binds the poly-histidine tag on our protein of interest.  Histidine is an amino acid that contains an imidazole group (a nitrogen and carbon containing ring).  Adding high levels of imidazole on its own outcompetes the binding of the his-tagged protein, thus making just the proteins legitimately affinity-bound to the beads elute off of them for analysis.  This, in combination with other changes, has been working well!

• Changing our epitope tag + affinity bead combination: remember how I mentioned that we’ve been getting the protein of interest off in the control lanes as well as the experimental?  Because it’s been coming off so strongly in those control lanes, we were wondering if it somehow magically had a poly-histidine tag on it, even though all notation about the strain says that it doesn’t.  So we sent it off for sequencing, and in the meantime created a new strain to use TALON affinity resin to pull on our putative target with the possible his-tag and hopefully co-purify Slx5 with a non-his tag.  This worked!  Well, Slx5 came down slightly in a control lane, but was much enriched in the experimental lane, which is decent evidence that they interact.  Especially because Slx5 is known to be a “sticky” protein (it non-specifically binds the TALON resin even when it doesn’t have the tag for it).  Incidentally, when the sequencing came back, it looked beautiful – in that the plasmid is exactly what we thought it was.  No his-tag.  Our theory is that the protein’s RING domain is binding the cobalt ions on the resin.  The RING domain is a zinc-binding domain, and I’ve found a couple mentions of it  being able to bind cobalt as well… which would explain a lot about its contamination in all of our previous control lanes.  It’s pretty frustrating, but also kind of amusing to look back on.  Actually, in writing this and looking back, I’ve come up with some ideas to try to reduce contamination in that control lane… too bad I’m at home and not in the lab (words of a true twamp, sigh).  Tricksy proteins…

Ubiquitylation Assays

So the preliminary run of this experiment worked – huzzah!  I love it when things in lab work relatively effortlessly; it is extremely rare.  Last I wrote, I had just potentially made a bunch of Slx5D strains that could be used to make the experiment even better, by comparing ubiquitylation levels in a wild-type vs. a Slx5D strain (the deletion strain shouldn’t show ubiquitylation on our target if Slx5 is controlling its ubiquitylation).  PS – that D in Slx5D is supposed to be the uppercase Greek letter delta, which won’t show up properly here.

After some grief, I finally confirmed the Slx5D strains by PCR and could start working with them.  Once again, the experiment worked wonderfully the first time around, which was amazing!  The wild-type background showed much more modification of our target protein, and the Slx5 background showed virtually no modification, despite the fact that tons of other proteins were still being ubiquitylated in that strain.  Just not ours, indicating that Slx5 does indeed ubiquitylate our target!  The only snag with this is that the way I  looked at the proteins didn’t isolate just my target and the ubiquitin, so theoretically the modification I see could be any modification, not just ubiquitylation.  To remedy this,  I tried some pulldowns to try to purify just our target and then probe for the ubiquitin to see if it is indeed ubiquitylated.  Unfortunately, this bit gave me a little more trouble (as pull-downs always do), and on the last day before I had to leave, I saw some preliminary good news that confirmed that I am pulling down our target, and it does have modification, and has more modification in the wild-type lane.  However, I still wasn’t able to re-probe again to look for the ubiquitin.  Nevertheless, I’m hopeful, and still just happy that the other part of the experiment looks so great!

So that’s where I left the summer with my big experiments, with relative successes in both!  Hooray!  A summary post will come shortly, where I’ll further sum up some of my findings and future directions for my thesis work.