The End

I’ve heard from so many people that somehow, magically, things just come together all at once at the end of your Ph.D., even if nothing has worked for years.  Of course, I didn’t believe them.

I was wrong.

In the past four months, I:

• submitted a manuscript
• had said manuscript accepted five days after submission
• wrote my dissertation, start to finish, in two weeks
• defended my dissertation
• graduated
• went on post-doc interviews
• accepted a post-doc position, after agonizing over my options for weeks

Suffice to say, it’s been a bit of a whirlwind these past few months, but, to quote a 90s staple, it finally seems like everything’s coming up Milhouse.

Beginning of the End?

Last week, I submitted my manuscript (fortunately, I was not doin' it wrong and received compliements from coauthors regarding how well it was written), officially received permission to start writing my dissertation, set my defense date, and discussed possible postdoc labs with my boss.

This week, I hide in an undisclosed location far away from the many, many distractions in lab and hunker down and write.  A completed draft of my dissertation is due on Thursday.

After 6 years and 8 months, there is finally, finally, a light at the end of the tunnel.  My escape plan is in motion.

Manuscript Writing

At my last committee meeting, at the beginning of the month, I presented my figures for my proposed manuscript to my committee and received the thumbs up to go ahead and start writing it (while finishing up a few loose ends and getting one additional experiment to hopefully work).  While seated around the table, my boss declared that he would like the draft of it by January 1st – exactly one month later.  The committee sat around joking that on the 31st, I’d be buying a plane ticket to California to get those extra three hours of writing in… and then hopping another plane to Hawaii to buy myself a few more hours… but oh, be careful and don’t cross the international date line!

I know that the comments were all in jest, a result of previous graduate students who were unable to meet deadlines and used all sorts of excuses.  But when I returned to the lab later and mentioned the deadline to other labmates, they all furrowed their brows and made comments about how a manuscript in a month is no easy feat.

Yesterday, two weeks before the imposed January 1st deadline, I emailed my boss my manuscript so he could take it on his trip to Europe (what better to do with 14 hours on a plane than edit it?!).  It took me a week to find the time between experiments to write a two page outline, and then four days to go from my outline to the finished draft, and one more day to get my figures polished.  I suspect the reason the writing went so quickly for me is because I have been working on this project for so long – I already knew everything there was to know about it, inside and out, so I did not have to waste time reading through lots of papers.  I also just gave my yearly data presentation to my department, half of which was on this project, and if I can stand up and speak about the project for 40 minutes, then I’d expect I could sit down and write about it as well.  However, I still can’t drop the nagging feeling that I’ve done something drastically wrong, since I finished writing the draft so quickly and with such ease.

Now the worst part: the wait.  Boss returns from Europe the end of next week.  Will my draft be returned with some minor edits, or a giant “MANUSCRIPT WRITING: UR DOIN’ IT WRONG!” scrawled across the top? 

Non-Academic Careers

I’m helping a post-doc in our lab take over the career development seminars for my program.  Our program really offers no support to students looking at options outside academia, and many of the investigators actively discourage trainees from pursuing other opportunities.   We are trying to help fill this gap by bringing in speakers from a variety of non-academic scientific careers to share a bit about what it is they do, how they got there, and give everyone some other career opportunities for down the road.
I’ve spent the past year or so conducting informational interviews with everyone and anyone who will sit down and talk to me about what they do, but unfortunately, I’m averaging only about a 10% success rate in regards to emails sent to actual phone conversations had.  I recently sent out another wave of emails to those in the area who were willing to talk to me about possibly coming in for a general career chat with others in my department, but I’m looking for more people and more careers to explore.
So, this is where you come in.  Throw those ideas at me – no matter how off the beaten path.  Once you have that Ph.D. in biomedical sciences, what can you do with it?  Super duper extra bonus points (and cookies sent in the mail) if you have an actual contact in the greater Washington area who might be willing to come talk to a bunch of senior graduate students and post-docs!

Time Warp

Frequently I find myself starting at my reflection in the mirror, unable to recognize myself anymore. The grey streaks of hair, the dark circles under my eyes – who is this person? Surely, I am not this old yet. It’s not that I find 27 to be old by any stretch of the imagination, but rather, my shock comes because it feels like time hasn’t moved over the past six years. Certainly, I am still just 21, fresh and wide-eyed out of undergrad, because I have nothing of accomplishment to indicate that six years have slipped past.

Looking around me, I accept that my friends are moving up in the world. They have new jobs they love, new promotions and pay raises, and are starting to accumulate the signs of achievement – purchasing a new car, moving to a better apartment or making the down payment on a house, having children. Friends who, in the amount of time I’ve been going into the same lab everyday and trying to force my protein to cooperate in the exact same assays, have held a job for several years, left to go back to graduate school, and returned to a different job. Friends who are already on child number two. Friends who discuss monetary investments and where to purchase property and retirement funds. And yet, there is nothing to indicate my passage of time. On the contrary, it’s like I’m moving backwards. I started out graduate school with a roommate. A few years later, I found myself married with a husband… who shortly left for residency in Philadelphia. I spent a year living on my own, and now find myself back to square one, living with a roommate, informing the surgeon’s receptionist that no, even though most individuals bring a spouse to the consult, I will be attending alone because for all intents and purposes, I am single once again (note: this has nothing at all to do with my husband not wanting to be there, but rather, the inability for him to take time off to travel to DC).

Husband’s best friend’s wife mentioned over a cookie baking session this fall that that they have decided to “pull the goalie.” A recent conversation with said best friend implied, though did not directly state, that his wife is pregnant. If it is the case, I am thrilled – overjoyed even – that they will be expanding their family. When I look at them, I see so many milestones of achievement. He went to graduate school and earned his first masters’ in New York City, then returned back to Philly, worked for a few years, and is now back in school again for a second masters’ degree. She has been through two lucrative jobs and recently left to start her own business as a personal chef and is thriving while completely loving what she does (a novel concept). Now, they are working on expanding their family. Both their lives are marked with achievements, personal and professional, indicating their successes over the past several years.

I look in the mirror and see grey streaks of hair and dark circles, but what I cannot see are the accomplishments that I find in all my friends. Six years into graduate school and I have no first author papers, and none coming up in the pipeline. I cannot even make a remote guess as to when I will graduate, other than to assure you that it will not be this May. Because I have no first author papers, I have no job prospects for after graduation. Not residing in the same state as my husband, there is no point in even discussing things like purchasing property or having children. I wonder, at which fork in the road did I make the wrong decision, and wind up stuck in this time warp of unachievement? Because certainly, a time warp must be the only explanation – I cannot possibly really be 27 and yet still be stuck exactly where I was at 21.

New Digs

What do you do when you don't have time to maintain the blog you already have?

You start writing for a second one, naturally!

I was so flattered when Brian over at LabSpaces invited me to blog for his new collection of science blogs that I just couldn't say no. But I also didn't want to give up my little home over here for the past five years, so I'll be writing both here and there.

So don't worry, no need to delete me from your RSS reader quite yet... just feel free to add on my second home, if you are so obliged! And look for things to pick up a bit around here too...

Come join me over at Disgruntled Julie's LaboRANTory!

Epiphany

Bright and early Sunday morning, as I was headed down to take dose my lab animals, I had an epiphany.

Remember back in elementary school when some classrooms had a “class pet” and students were able to take the [rabbit/guinea pig/hamster/other animal in a cage] home sometimes over the weekend?

What if the same could be done with lab critters?

Friday night comes, and you stack up all the cages, pack the animal toolbox up with syringes, needles, alcohol swabs, calipers, drug, and whatever else you need…. and take your work home with you.  Then, instead of the weekend morning ritual (alarm goes off obnoxiously early… nearly throw phone across room in frustration… lie in bed thinking about how much you hate the lab… how much you hate your animals… how much you hate your life… how much you hate that you are the only person in the lab stuck with daily dosing as opposed to M/W/F… how much you hate that your PI always talks about how he always took the Sabbath off during his research fellowship but doesn’t extend you the same courtesy… hate that you are the only one in the lab in a long-distance marriage yet never get to sleep in with your husband on the 2-4 days per month he comes to visit… hate your PI… etc etc etc), you could climb out of bed, quietly shuffle out to the living room, get yourself situated, dose your animals from the comfort of your pajamas, and then climb right back into bed.

I really think I’m on to something here.  I’m going to be starting another much larger study pretty soon, so maybe I’ll use this as a bargaining tactic.  “Why sure, PI, I’d be delighted to come orally doze a bazillion mice every day so you can get the drug into clinical trials ASAP… as long as I don’t have to physically go in on weekends to do it!”

Disgruntled Julie: A True Hero

This evening, I was just minding my own business at home when I heard a blood-curdling shriek come from across the hall, causing me to immediately jump up and make sure everyone was okay.

(Weeeellll…. kind of.  I had just finished elliptical-ing, and one of the big benefits to owning your own elliptical is that no one sees you while you work out, so first I had to put on a shirt, and then I cracked my door open a few millimeters to stick out an eyeball to make sure the screams weren’t because an axe murderer was lurking in my building.  Then I tentatively took the few steps across the hall, leaving my door open and my body pointing back towards my door, in the event said axe murderer answered, in hopes that I’d be able to bolt back to the safety of my own unit and slam the door in his face.)

My across-the-hall neighbor opened the door, pale and shaking.  “Oh (gasp) my (pant) G-d (pant, gasp) it’s (pant, pant, gasp)…. a (pant, pant, gasp)… (pant, pant, gasp, pant, building up the suspense….) MOUSE!”

Dun DA DUN!  SCIENTIST TO THE RESCUE!

I cornered the mouse, trapped him on a dustpan, and picked him up by the tail to carry him outside where he scampered off to freedom (and by scampered to freedom, I probably mean someone else’s apartment, where he may meet his ultimate demise via a mousetrap).

On my way out, my neighbor caught her breath enough to tell me that I was her hero.

That’s right.  Disgruntled Julie: Saving Single DC Women from Eenie, Weenie Terrifying Mice.

Well, at least one technique from the lab is useful in a real world setting.

(I really don’t understand why people are so terrified of mice.  Does she look vicious?)

Daylight Savings Proposal

I’m not a fan of Daylight Savings Time to begin with… I think it’s a bit outdated.  After all, most of us are not farmers and do have electricity, so the need to push back the time in the fall to gain light an hour earlier isn’t the necessity of years past.  And as much as everyone enjoys gaining an hour in the fall, springing forward is a real pain.

I don’t think the government is going to go along with my elimination of DST, so I have a suggestion.

How about, instead of 2am Sunday morning, we end DST at 10am Monday morning?

We assemble into lab meeting and start discussing general lab business.  At 9:59, the person presenting for the meeting stands up to talk, opens their powerpoint presentation… and suddenly it becomes 11:00am.  Lab meeting over, everyone dismissed.

I wouldn’t mind if that hour disappeared from my life…

Boston Recommendations

Later this month, I will be heading up to Boston for the AACR Molecular Targets & Cancer Therapeutics meeting.  It’s been quite a while since I’ve been to Boston – the last time was the summer between graduating high school and starting college!  I’ll be in the Back Bay area, so if anyone has any suggestions for things to do in the evening (or during less interesting sessions!) or places I should eat, let me know!

Also, if anyone else is either attending the conference or a permanent resident of Boston, send me an email if you are interested in perhaps trying to meet up while I am there!

Harumph

I have things I’ve been meaning to post about, but instead, in light of my current situation, I leave you with this:

Do you see the giant cavernous slump in 5th year? That is exactly where I am right now. And right at the very lowest point, it should say “All experiments that were working have suddenly inexplicitly stopped despite weeks worth of troubleshooting”.

I have fallen into the Fifth Year Pit of Despair.

Even one of the PIs of the lab recognized the Pit of Despair, acknowledged that no one could possibly be motivated in this situation, and told me to take the rest of the day off and eat ice cream. And who I would I be to argue? (Okay, well, instead of ice cream I had non-dairy, fruit-sweetened Soy Delicious, which is possibly my Favorite Thing Ever, but same idea.)

Anyone have some rope to start dragging me up the giant peak of motivation to lead towards my defense?

***UPDATE***

One of my favorite Twitter followers, @bam294, who always provides a hilarious PI-point of view of situations, sent me this SNL clip to describe my current situation.  Indeed, there was a light at the end of the tunnel… but… alas…. it’s broken.  I guess I just need to “fix it”. (Fast forward to ~2:05.)

Three Steps Forward, Two Steps Back

Fourth grade was a pretty crappy year for me. My teacher was simultaneously going through a bitter divorce when she learned that her teenage daughter was pregnant. She chose to take out her frustrations on us, yelling, screaming, and teaching us that no matter what went wrong in life, it was all our fault. Not caring enough to take any responsibility for her students, her answer for the somewhat disruptive class clown was to sit him next to me, and tell me I had to control him… and if he misbehaved, it was a bad refection on me. Practically every day, he would lean back on his two chair legs, the teacher would yell at him, and eventually take away his chair and force him to stand for the rest of class. Inevitably, he would wait until I was concentrated hard on reading, walk up behind me, yank the chair out from under me, and dump me on the floor to steal my chair. And, everyday, the teacher would tell me it was my fault for not doing a good job of controlling him, so my punishment was that I had to stand while he had to sit.

The one bright spot in the year was my gifted education. Unlike many places, like my husband’s school district, we did not have one completely separate class for gifted. Instead, those in the gifted program just left their regular classrooms for an hour per day. There were three of us from my classroom in the gifted program, and we were all having an equally miserable year. In an effort to drag out the time until returning back to our regular class, we used to walk down the hallway taking three steps forward, two steps backward, essentially progressing forward one step for every five steps taken.

Three steps forward, two steps back seems like the best description of my research right now. Remember the impossible purification I wrote about a few weeks ago? Well… it turned out to not me so impossible after all. One night, lying in bed unable to sleep, I had an idea… a hunch that I thought just might work. The next morning, I told my PI of my plan, to which he replied, “It won’t work. Don’t bother trying.” This statement came from the same individual who just a week before went on and on at my committee meeting about how I was a better biochemist now and had surpassed his knowledge of purification, so surely I could find a way to purify this protein. Rather than get (too) disgruntled and annoyed, I went with my gut and attempted it anyway, and two days later, smugly shoved a coomassie under his nose showing a dirty start lane and two perfectly purified bands in two separate fractions. Three celebratory steps forward!

After showing that Protein Y had properly refolded (the purification involved denaturing the protein and refolding it on the column) and still bound to Protein X, PI said the worst possible statement. “Next,” he said excitedly, “all you have to do is crosslink the lysines and that will be easy!” You see, it’s like sports. The second an announcer makes some comment like “[Quarterback] has not thrown an interception in 612 passes!” it automatically, no-fail, means that the next pass is going to be intercepted. Always. And this rule applies to PIs as well. The second he opened his mouth and described this step as “easy”, I knew I had it coming.

As it so turns out, the family of crosslinkers that will connect these proteins in the way that we need them to be connected works through linking the primary amines in the side chain of lysines. What this means is that you cannot have any amines in the buffers, because it will wind up crosslinking the buffer component rather than the proteins. And do you know what imidazole contains? Secondary amines. Harumph. So, I called up tech support at Pierce and the conversation went a little something like this.

Disgruntled Julie: Do secondary amines affect the crosslinking?
Tech Support: Yes, you can have tertiary amines, but not primary or secondary.
DJ: Is there anything I can do to get around imiazole in the buffer?
TS: Dialyze out the imidazole!
DJ: I’ve spent three years already trying to do that. Not going to happen.
TS: Have you tried doing it in steps?
DJ: 4 step, 6 step, and 8 step. Protein precipitates.
TS: How about overnight?
DJ: Precipitation.
TS: With glycerol?
DJ: With and without.
TS: How much glycerol?
DJ: 10%, 20%, 30%
TS: Spin column?
DJ: Precipitates.
TS: Desalting column?
DJ: Precipitates.
TS: Well, shit.

Indeed. My sentiments exactly. I finally have both proteins purified, and I cannot crosslink them because I absolutely, positively have not been able to dialyze the imidazole out of the protein.

O n e. T w o. Two giant steps backward.

Isn’t It Ironic?

I just happened to notice that my post yesterday on my protein purification woes drew Millipore as an advertising sponsor.  Obviously, this makes sense – I talk protein science, scientists come and read, a life science research biotech company sees a target audience.

But the irony comes in the particular product which Millipore is toting – a 0.5mL centrifugal filter for concentrating or desalting/buffer exchange of proteins.

Have I mentioned that I have literally spent 3 years (do you see a trend in my never-ending, unsolvable research problems) attempting to dialyze imidazole out of Protein X?  And how many Millipore columns I have used?

Millipore, why must you taunt me so?  What’s next, Pierce advertising their Slide-a-Lyzer dialysis cassettes for quick and easy buffer exchange? (I am pretty sure that I am single-handedly keeping Pierce in business with the number of dialysis cassettes I use for all my unsuccessful dialysis attempts.)

New rule: if you advertise something as “delivers great performance”, then you better come to my lab and make it work with MY protein!

Impossibilities

Last week, I had a committee meeting.  In preparation for the meeting, I met with my PI to discuss how to rework my aims, change my hypothesis, and the best way to lay out the “future manuscript” my committee likes to see (i.e., when I acquire data, I will put it into a paper like so).  This lead to a very long discussion in which my PI decided that I had too much chemistry in my project (too MUCH chemistry?  blasphemy!  there is no such thing.) and wanted to try to shift it back to the biochemistry/biophysics side of things.

Now, let’s backtrack a wee bit here.  My second and third years of graduate school were encompassed mostly by failed purification attempts of a fragment of a particular protein which binds to the main protein studied by the lab.  Let’s call this protein fragment Protein Y.  Now, I was not the first person to be assigned the task of purification; rather, a post-doc in the lab and our protein purification lab tech had each sacrificed months of their time in lab attempting to purify Protein Y with no success.  I, however, was not aware of the previous failed attempts, and presuming Protein Y to be like any other protein for which I found a purification strategy, forged ahead with elaborate plans of what to do with said protein, including crosslinking and mass spec to identify the region where Protein Y binds to Protein X, and attempts at co-crystallzation, thinking that perhaps Protein Y would stabilize Protein X enough to form crystals.

I then spent two miserable years trying to purify protein Y, with no success at all.  I certainly do not consider this time to be a waste… rather, I acquired so much knowledge about protein purification and various ways to purify a protein that I have now become the resident protein purification expert.  The chair of biochemistry has informed me that there is quite the demand for biochemist post-docs with expertise in protein purification, so even though the project tanked, I still think it was integral to my future purification success and perhaps even obtaining a future position.

The Protein Y Purification Project never officially closed, but rather, my time was shifted to working on other projects to help get a paper out the door.  I, naively perhaps, assumed that since my thesis aims had shifted to reflect new developments in the lab, and a project which interested me much more (more chemistry! more biophysics!), I was off the hook from my dreaded Protein Y purification days.

Until last week, when I met with my PI.  He wants me to go back to Protein Y purification, and re-visit the crosslinking and co-crystallization ideas.  Nevermind that the lab has now spent 3+ years trying to purify the protein, racking the brains of one post-doc, one protein guru technician, and one very disgruntled graduate student.  I moaned.  I groaned.  And boy, did I complain.  And then, I offered up a perfectly acceptable solution for determining the binding location without having to purify the protein.  My PI thought about this, and agreed that it would be a good alternative… but really, he’d rather just have me purify the protein.  I have no doubt that this has less to do with my own experiments, and more to do with all the things he thinks he could do with a purified protein after I leave… after all, his priority is the future of the lab, and not getting me out the door in a timely fashion.

At my committee meeting, after I presented everything (quite well, according to my PI), the chair of my committee asked what I thought the biggest challenge would be to completing my aims.  Without hesitation, I pointed directly to Protein Y as the source of my likely shortcomings.  The other members of my committee nodded and agreed, after witnessing two years of my failed attempts.  But, never one to be discouraged or accept that perhaps a protein just cannot be purified, my PI immediately jumped up and went through a spiel about how I am a better biochemist now than I was two years ago and how I will certainly have much more success this time.

After just one more week back with Protein Y, I already want to strangle myself.  I wonder how many more years I will have to spend on this before my PI accepts that maybe, just maybe, my research is impossible.

Non-Graduation

I would need a third hand (maybe a fourth) to have enough fingers to count off the number of times some smug old jerk of a doctor has made a comment that the reason I am in research pursuing a Ph.D. is because I am clearly not intelligent enough to be a “real” doctor.  Of course, we all know that this is utter BS – but I also know that there is no point in trying to change the opinion of a old, ignorant, pompous man, so I just let it all go in one and ear and out the other.  Interestingly, my PI, who is an MD (aka, a “real” doctor), has mentioned on multiple occasions how he believes the Ph.D. process is exceedingly more difficult than medical school (and has unofficially mentioned to a few undergrads that if they are interested in a clinically based research field, like oncology, where physician-scientists are far more respected than in a truly basic science… they will have a much easier time just going through medical school and doing research afterward, even if they never intend to practice clinical medicine). 

While medical school may be more intense as far as the number of hours (while I have spent many a night working overnight in the lab… it pales in comparison to Husband’s call nights as a medical student), obtaining the Ph.D. is the marathon in comparison to the sprint, and the truly difficult part is not knowing when (or if?) the race will ever end.  It’s not just showing up everyday, studying, and working hard – it’s having to discover something brand new in your field, which is then important enough to warrant publication in a decent journal; often, multiple times before you are permitted to even start writing.  So while medical school and graduate school are entirely different beasts, I’ve always thought it was absurd for anyone to consider one entire population – MDs versus PhDs – to be more intelligent than the other.

Until now.  It recently dawned on me that had I attended medical school, I would have graduated this past weekend.  As in, over.  Finished.  Done.  Dr. Disgruntled Julie.  Four years and out.  The day my husband started his first year medical school orientation, he also knew his graduation date.  But… instead, I went the research route, via a Ph.D.  I could have graduated this weekend, but I did not.  Instead, I have another 2?  3?  12? years to go.

Degree FAIL.  I have seen the light.  MDs are FAR wiser than PhDs.  Lucky bastards.  Oh wait, excuse me.  Lucky INTELLIGENT bastards.

Diary of a Doctor’s Wife, Episode 005

I certainly cannot attest for all marriages, but ours seems to be one based on double-standards of doctoring. The frustrating double-standard of the moment deal with the Rules of Sickness.

The Rules of Sickness are as follows:

1. When Julie is sick, she must sleep on the couch and avoid the bedroom at all costs, as not to infect Husband with germs, because he is a Doctor and is therefore Very Important and Saves Lives, and cannot afford to get sick.
2. No matter how sick Julie is feeling, she is still expected to cook dinner for Husband, because he is a Doctor and therefore works Very Hard at Saving Lives, and comes home hungry and tired and needs to replenish his energy.
3. When Husband is sick, he still gets to sleep in the bed, because he is a Doctor and works Very Hard and needs a Good Night’s Sleep and therefore should never have to sleep on the couch.

As a result of these rules, 9 times out of 10, when I am sick, it does not spread to Husband (fortunately for him), and 9 times out of 10 when he is sick, it does spread to me (unfortunately for me).

At this point, if you are like my parents, my mother-in-law, my labmates, or any of my friends, you might be thinking to yourself, “Well, at least your husband is a doctor; surely, when you are sick, it is useful to have a doctor to take care of you!”

Rule #4: Husband is a Doctor and works Very Hard all day taking care of Real Patients in the hospital and therefore has No Patience to come home and take care of Wife-Patient.

So, in other words, being married to a doctor makes me more likely to catch whatever germs he brings home from the hospital, and less likely to have someone who remotely cares that I have become sick. It’s totally a win-win situation.

And, in other humorous news, I’ve gone from raspy nails-on-a-chalkboard voice to no voice at all. And today, my PI asked me to call around and get price quotes on both a fluorescent plate reader and an ultrasonic bath. At the end of the day when I hadn’t done it, he was obviously quite visibly annoyed. Er, I have no voice – am I supposed to try to set up an iChat session with our Fisher representative and mime out “ultrasonic bath”?! Come to think of it, I was always pretty good at Charades…

WARNING! THIS IS AN EMERGENCY!

This morning, my PI declared that I needed to do “emergency CD”.

Quick!  Somebody sound the biochemistry emergency siren!  This is not a drill!  This is an emergency!  A CIRCULAR DICHROISM emergency! 
WOOOOOOOOOOOooooooooooooooooooOOOOOOOOOOOOOOooooooooooooooooOOOOOOOooooooooooooooooooooOOOOOOOOOOoooooooooooooooooOOOOOOOOOOOOOOOOOOooooooooooooooooooooooooooooOOOOOOOOOOOOOOOOooooooooooooooooooo!  
This is not a drill!  This is an emergency!  Biochemistry emergency!  Proceed to preassigned emergency station!

Now, I understood his point.  In light of the ugly CD data I generated earlier in the week, I tried dialyzing out the EDTA, but (much like what happens with other attempts to dialyze the protein, when it chooses not to precipitate) running on a gel afterward resulted in no visible protein in the post-dialysis lane in comparison to the pre-dialysis lane.   Not one to ever be discouraged by something like the inability to see protein stained on a coomassie gel, he declared that I immediately had to hop on and see if I could get a spectrum before I left, because the protein degrades so quickly that it would no longer be worthwhile by the time I returned at the end of the weekend.  Hence, the “emergency” situation.

While everyone else in the lab snickered at the thought that circular dichroism had suddenly become an emergency, my PI stood behind me while I called up the Chair of Biochemistry to see if if the spectropolarimeter was available for my use (seeing as how I am pretty much the only one doing CD, the bigger question is always if I can track down the Chair to get the key to the room). 

While I was doing the “emergency” CD, I was once again GChatting with said Best Friend who sent the previously posted Calvin & Hobbes cartoon, relaying the story of my morning “emergency.”  He asked if biochemists had a universal distress signal – you know, shining a double helix in the air, like a Batman signal.  Once Operation Emergency CD was complete, all units were given the clear to stand down, and thankfully no one (aside from the nitrogen tanks) were required to shelter in place.  As I was about to sign off, Best Friend referred to me by my apparent new Superhero name…. CD-Woman.

So, if you happen to see someone dashing to your neighborhood spectropolarimeter, ice bucket in one hand, 1mm cell and pipette in the other, wearing a lab coat and undies on the outside (because naturally, that is the mark of a true superhero)… wish me luck.  I’m off to save the world from another circular dichroism emergency situation.

Bang Head Here

Today, I needed this hung on the wall next to my lab bench:

I’ll spare you all the moany, groany, whiney in-depth details, but suffice to say, my World’s Worst Protein is, once again, living up to it’s uncooperative title. I have spent the past 5 months trying to move forward with circular dichroism by dialyzing out the imidazole following elution (unsuccessful – always precipitates), dropping the pH (unsuccessful, still precipitates, and dropping it too low unfolds what has been refolded) and attempting to elute with EDTA. Finally (finally!) I was able to get the protein a) pure enough, b) refolded on the column, and c) off the column with EDTA, and today was the big reveal.

Suffice to say from the previous “Bang Head Here”, you can probably guess that rather than a chorus of angels descending from the heavens singing as a beautiful spectrum appeared as the result of all my hard work, thereby bringing me one step closer to maybe, possibly, potentially, someday graduating… I got an ugly bunch of sharp zig-zagging lines, because the amount of EDTA it takes to get my protein off the column apparently causes significant light scattering. Damn.

Nothing like watching 5 months of work unravel in 2 minutes. Bang head here, indeed.

Of course, while watching my spectra unfold in real-time, I happened to be Google Chatting with my best friend, which may have involved some explicit language and a pity-party for one regarding my inability to get just one decent looking spectra for Protein X. Within 3 seconds of the use of my word “spectra”, the followed magically appeared in my inbox from said best friend:

Yes, the “spectra” in reference is totally different, but the theory is the same. Learn more about the structure of the protein, and get a publication… and, while I can’t say it’s in my short-term plan like Calvin, I certainly wouldn’t turn down the Nobel Prize, the “get rich” money, or the talk show invitations. But Hobbes, I hate to break it to you – I don’t think scientists get too many babes.

P.S. Dear Protein, I hate you.

Ph.D. in Cosby-ology?

Since Bill Cosby was born and raised in Philadelphia, my husband immediately jumped on The Cosby Show bandwagon as a child (I don’t understand the instant appeal, but somehow, if it has anything to do with Philadelphia, no matter what it is, it automatically means that it is amazing and wonderful and fantastic according to my husband).  I’m not entirely sure under what rock I was hiding between the years 1984 (okay fine, I was a mere 6 months old when the show first aired) through 1992, but I never once saw a single episode throughout my entire childhood, a fact which appalls my husband.  Feeling the need to remedy this situation, every time I am home between the hours of 4pm – 5pm from now on, he ties me down to the couch, and forces me to watch reruns on our local WGN station.

As I’ve been home sick with a high fever all week, and my husband is working 10 hour block ER shifts which currently happen to leave him at home in mid-afternoon (thus meaning he is around to play King of the Remote at 4pm), it’s been 4:00 Cosby Hour all week.  Today, while rolling my eyes at some situation (or perhaps at Dr. Huxtable’s ridiculous sweater-of-the-day), Husband told me I shouldn’t make fun of someone who had worked just as hard as me educationally, since Bill Cosby has a Ph.D.

I tried to call him out on this, because I had heard that Bill Cosby had failed out of high school.  A Google-search revealed that he actually has his Ed.D., and the title of his thesis was:

An Integration of the Visual Media Via "Fat Albert And The Cosby Kids" Into the Elementary School Curriculum as a Teaching Aid and Vehicle to Achieve Increased Learning

Yes, Husband, yes.  I am quite sure that Dr. Cosby has worked every bit as hard as I have in his educational pursuits.  Now, how can I work my week of laying on the couch watching The Cosby Show into my dissertation?

Title My Talk!

In our program, every year each Ph.D. student must present their data in an hour-long talk to the entire department. I always kind of figured this was standard practice in graduate school, though in talking to students in other programs at this university and those at other universities altogether, it seems like the majority never give a talk to the entire department until their thesis defense. Personally, I like the way we do things here – it’s a great way to practice giving talks in front of an audience, keeps everyone abreast of your research, and often results in great suggestions from PIs outside your thesis committee which you may otherwise have never received.

I may, however, feel this way because I have zero problems with standing in front of a room and talking, unlike the standard stereotypical scientist. Plus, even though I generally don’t have much data to share (who wants to look through 500 coomassies that show my inability to purify a particular protein?) the techniques I use are completely different from others in my program, so by default, my talk is always new and interesting, which forces people to pay attention. My program is in cancer biology, rather than biochemistry or pharmacology or structural biology, so we are tied together by the fact that we study cancer, not by the way in which we study cancer. 98% of the program studies cell signaling and knockdown genes in cells and in animals, and while they produce some hot science and generate some pretty cool results, the techniques they use are all same old, same old in so much as we see the same thing every week.

Then I come along, and my results come from surface plasmon resonance and circular dichroism and fluorescence polarization, and I talk about things like molecular modeling with structures from co-crystallization. While nobody would ever stop and explain how to “read” a Western or a Kaplan-Meyer plot, I can’t exactly put up a CD spectra and make a statement about the structure of the protein without taking the time to explain a little about CD, or throw up a graph with polarization that shows that my small molecule displaces a peptide without a few cartoons showing how FP works. That’s what I love about my project – even though it’s taking me a long time to generate worthwhile data, my presentations are always interesting, because I am the black sheep of the program doing something COMPLETELY different than everyone else (all biochemistry/biophysics), and people are interested to learn about what it is that I am doing.

That said, tomorrow I give my once yearly talk, and happen to also be going for the double-whammy and also having a thesis committee meeting (it is not, however, standard to have committee meetings every three months at this university… the program asks for every 6 months, most people aim for once a year, and I obviously pick up their slack by squeezing in extra meetings in the middle). So, as I sit here looking over my slides for tomorrow’s presentations, I find myself unsure what to title my talk.

Should I opt for:
(a) 1001 Ways Not To Purify Protein X
(b) 50 Unsuitable Buffers for Circular Dichroism of Protein Y
(c) Protein X and Protein Y Interact But You Probably Don’t Care, and I Really Don’t Either
or
(d) Protein Y Isn’t Improperly Folded, It’s Simply Structurally Ambivalent

Other suggestions welcome.

Also, since Protein X is a helicase, I think I need to fit this somewhere in my talk: