A Rant about Western Blots

I hate Western blots.  I think they are the most annoying, frustrating, and obnoxious experiments to do in lab.  I hate that the process takes at least two days and that it's so antibody-dependent.

First, I am fully willing to admit that I am not good at Western blotting.  My films usually look a little messy, and I've taken several steps to clean up my methods to no avail.  The process is so touchy and there are so many steps that, apparently, I have trouble performing properly.  That's my problem though.

What I hate about Westerns is how subjective they are.  One antibody uses a milk block; another uses BSA; the last one takes three quarts of finely grated basil and a dash of kosher salt.  The first one needs to be incubated overnight at 4 degrees; the second needs three hours at room temperature; the third needs to be set on a wooden stool in the midday sun from exactly noon to 2:30 PM.

The "voodoo" science involved in making these antibodies work bothers me.

It also doesn't help that the department's developer was made prior to the Cold War era (and I'm probably not even exaggerating too much).

What alternative is there?  None.  I may hate the process, but there's no way I'm getting around doing Western blots for the rest of my laboratory career.

Science music!

I like listening to music a lot, and I always find it comforting to listen to it in lab.  This often leads to me basically choosing what everyone else has to hear... which was fine when there were two people in the lab.  Now that we've grown in size, however, I feel bad about making everyone listen to my music.

In reality, they have every right to plug their iPods into the stereo or choose a different radio station... but they don't.  So I do.  Does this make me a bad person... because I listen to my music all the time in the lab?  Maybe I would feel bad about it if anyone else expressed interest in playing their music.

I guess that I'll just continue listening to Britney Spears and the Spice Girls on a regular basis.  Nobody's complained yet...

My lab playlist:

• Joss Stone
• Girls Aloud
• Maroon5
• Clarika
• Britney
• The Saturdays
• Janelle Monae
• Amy Winehouse
• Nicki Minaj
• Ke$ha
• Erykah Badu
• MGMT
• Paula Abdul
• Spice Girls
• Alicia Keys
• TLC

I'm not entirely sure why they're almost all female singers, but I guess that's my preference.  I like to think that I have a mixture of music, but I tend toward radio-friendly pop music.

Pressing the "Submit" button

As I mentioned in the last post I did, my PI is gone on vacation and I've been working my butt off with a number of different experiments.  Today, I finish the last experiment we need to submit our latest paper.  The problem is that she's out of the country... leaving me to submit the paper.

She sure didn't seem to have any problems with me submitting the paper.  Maybe I should take that as a sign that she trusts me to do this on my own...  But it's still freaking me out.  What if I upload the wrong file?  Did I choose the right keywords?  Is my new figure "pretty" enough?  I shouldn't be worried since it's not an enormous paper, and it's not going to a super-high impact journal.  Still, it's my first time...

Submitting this paper is scary and fun at the same time.  I guess it's a good experience for the future, regardless of how I feel about it.

While the boss is away...

My PI is on vacation to a far-away land.  And I am not.

As usual, when the boss is away, everyone in the lab is even more stressed than ever.  I'm still trying to finish up the comments on the paper we submitted last month, so I'm under a deadline to submit it before vacation's end.  Everything would have been done last week had I properly read the labels on our antibodies.

Dammit.

But all is not lost - I will finish my experiments this week, get back on track, submit the paper, and maybe even relax ever so slightly.

On a positive note: the experiment I so desperately needed to work for my thesis actually worked.  The universe doesn't hate me!  And the best thing is that I included almost every single possible control in my first set of experiments.  Now all of my data is organized, beautiful, and figure-ready.  I've never had my experiments work so beautifully on the first go.

I could get used to that.

Oh, and I changed the background on the blog because I was playing with Illustrator and liked what I made. :)

No time for sickness

The Independence Day weekend was fantastic, visiting my undergraduate school and bumming around town during the hot, sunny, beautiful summer days.  At times I miss being an undergraduate, particularly in the summer, when I had few responsibilities and I had a lot of free time to just relax.  Those days are apparently over, and while I might reminisce, I feel that it is truly time to move on and be a "responsible" and "mature" graduate student.

And while on that mini-vacation, I became sick.  It started just as a sore throat, and now it's going into full-blown sinus nastiness.  I can't breathe and my head feels like it's stuff in a giant feather pillow.  It's not like I'm dying, but I'm not enjoying this current state either.

Regardless, I don't really feel like working or thinking today: my head is tired, I feel sick, and my vacation days just ended.  I would like to crawl into a small, science-less ball and take a nap.  It's not like I'm asking for much.

But no. Cells must be split, infections must be inoculated, plasmids must be digested, and primers must be ordered.

I'm just going to do it all slowly.

Paper reviews are in!

My first paper from my graduate lab is moving along well.  We submitted in April and have just now received the reviews back.

Impression thus far: not too shabby.

Overall, the reviews were very fair, which is something I've come to appreciate after my first experience with paper reviewers.  There is no point at which I'm surprised at what the reviewers wrote, and I feel that the changes they propose will only help the manuscript.

One of the big experiments that one of the reviewers so cleverly proposed we perform: done.  Last December.  I guess that I was thinking ahead when I did that experiment!

I'm liking this summer a lot so far.

Finally, Science is working again

I had been complaining time and time again about how nothing I touched would work.

It appears that my luck has changed.

Nearly every experiment I performed last week worked miraculously.  My PI and I have decided to expand a manuscript that we recently submitted, and she is all for me cranking out the data.  Surprisingly, my cells and antibodies have decided to cooperate with the endeavor, and my numbers look fantastic.

Just yesterday, however, I realized how crazy busy this week has been.  I've been trying to finish up the data for this paper (which is going to take a couple more weeks...), continue working on my core thesis proposal data, figure out what the heck is going on with the mice we just received from another lab, optimize some new reagents, and help and out the new graduate student.  And then my PI suggests I start another project with the new student.  Another project.  On a topic that I have never even thought about.

I've got my core thesis project, which is rolling along nicely.  In the last week, I've finally started working on all three aims in tandem, meaning that things are starting to come together at least a little bit, but it's overwhelming sometimes.  Additionally, I've got another project that involves figuring out what this unknown protein does (fun but overwhelming sometimes).  Add that on top of the collaboration we have with another lab, the data for which I am solely responsible.

And now another project.

I am starting to realize that I'm only one person and that there simply are not enough hours in the day and there is not enough caffeine in the coffee for me to accomplish all of this.  I simply don't have the hands to perform seven different experiments at once.  Over the last year, I have mastered the art of multitasking in the lab.  On any given day, I will, without a doubt, be performing at least five different experiments with five different sets of equipment.  But at the same time, it's hard to just throw some more projects on top of the pile.

Ugh.  I talked to the new student about it, and she said that my PI wouldn't have given me all of this to do if she didn't think I could do it.  A little part of me wants to believe that...

Or maybe I should just stop complaining (and typing this blog post) and get my butt back to work... :)

Immunology scares me

Working in a virology lab, I should have known what I was getting into.  Prior to joining the lab, my PI told me that she expected all of her students to learn immunology, whether that be through taking the painful class or through osmosis and reading.  Because I do not want to take this painful class, I have elected to read the Janeway Immunobiology textbook and learn as much as I can by teaching myself.  So far, so good.  Nonetheless, I can't help but be overwhelmed by immunology.

I think it stems back to my undergrad days, when very few people in my major (genetics and *cough* math) took the immunology course.  I knew of people that took it and enjoyed it, but it was just kind of ignored by most people with my major.  Thus, I've never really been exposed to immunology in any respect.  Combining that with the intense immunology talks that I've attended over the last couple years, it seems very intimidating.  And for many reasons:

1) Why are there so many types of T cells?!
2) Are those FACS plots really just the researchers making dots on a chart?
3) What are all those CDs?
4) Exactly what language are immunologists speaking?

Of course, this isn't a complete list.  I could go on and on about why I think immunology is scary.

I hope that in the future, I will look back on this post and think about how far I've come in learning immunology.  I do realize its importance, but it's just so difficult for me to learn for some reason...

Cloning is fake

My electron micrograph of E.coli,
courtesy of Adobe Illustrator

I've been trying to clone a set of 9 genes for the last three months.  Three months.  Not only is this ridiculous and a waste of my time, it is frustrating and makes me angry.  I received the vector from the lab across the hall, performed my PCR and tried to pop that insert into the vector.  You would think easy-peasy, but not so...  Even my PI is getting frustrated with it, and she's not even doing the cloning.

I complained about this to one of my friends in a neighboring lab, and she joked that the solution is simple: "Cloning is fake. It's all made up in order to make you feel like a failure."

While both of us know full-well that cloning, indeed, is not fake, I am going to use this as my excuse for the meantime... at least while my vector refuses to digest, ligate, and replicate.

Is that so much to ask?

Update: Clones 1, 2, and 3 are complete (Yes!).  Now on to 4 and 5 and ...  Soooo close...

The Palin Emails

Oh you betcha.  Former Governor Sarah Palin's emails are let loose upon the world and people are searching through them for juicy little tidbits at this very moment.  Those emails must be mighty interesting for people to spend so much time reading and analyzing them.

Which makes me wonder...

If I release all of my emails to the public, will some group of people read and analyze them for me?  Wouldn't it be wonderful to have all of my daily emails summarized for me?  

It would make my day a little bit easier if I didn't have to respond to emails too...

Just saying...

Department seminars: Oh, the joy

I gave my first department-wide presentation on Friday.  Granted, it was a short, 30-minute presentation that mostly focused on me introducing my project and describing the thinking behind it.  Nonetheless, it was still exhilarating and tiring at the same time.  When it was over, I was tired - even more when I returned home tonight.

I suppose I wasn't too worried about the presentation, as it is not very high-pressure.  Not too many people showed up, as it is summer and attendance tends to dwindle after eight months of these presentations.  Still, it was nice to see the 20-30-some people listening to me talk about what I love to do.

For the most part, I was able to answer questions reasonably well, and I also gained some suggestions that I think should make my research stronger.  At least I hope.

One thing that I certainly need to get over is my sensitivity toward criticism.  The data I presented was quite solid: I did not include any controversial data or anything that I wasn't absolutely certain is true.  I've learned from my previous, smaller presentations that I'm just not good with handling criticism.  When someone starts to evaluate my data critically, I usually become very defensive and feel as though I'm being attacked, which certainly isn't true in all cases.  Now more than ever, I need to get over my sensitivity...  It's going to get me no where in science.

But now my department-wide presentation is done for a year.  Next up: committee meetings. Joy.

The most frustrating part...

I hate office politics.  I hate when these politics are distracting and needless.  I hate that if I don't send the right email at the right time with the right words in the right order, I may have offended someone.

I've been thinking about this a lot lately, mostly because I've gotten some heat for writing an email and not being appropriate (apparently).  I certainly don't know who I could have offended, but I suppose if my email was taken the wrong way, maybe it could have been misconstrued.  In reality, this is ridiculous, and I shouldn't be wasting my time even thinking about the repercussions of saying "Hi" in an email versus "Good Morning" or "Hello."  Nor should anyone waste time being offended by my improper greeting.

But alas, I'm even writing a blog post about it.

From now on, I'm going to start all my emails with one of the following:

1) Yo
2) What's up
3) Heeeeeyyyy (for more formal situations)

This will solve all problems.  Certainly.

Am I not in on a joke?

I use Google Reader to stay abreast of the goings on in my favorite scientific journals.  When going through my feed, I sometimes come across journal articles that boggle my mind, usually from one or two specific journals (which will remain anonymous in this post, though your guess is probably right).  My initial hint that I should immediately skip over a paper is if there is a question mark in the title.  The first rule I learned when starting to write in a scientific manner is that one must never, never use a question as a title.  Instead, one must convey their findings in an articulate statement.  As I was taught, making your title a question was "the easy way out" and was discouraged.

But I digress...  Some of the papers that I come across (and I see mocked on Twitter) just seem like jokes.  I tell myself that there must be some scientific reasoning behind the hypothesis in the study (if there is one) and that I'm just not up-to-date in the field to know what's important.  Still, there are other moments when I'm just rather awe-struck at what has been published.

One article that I remember was retracted due to the controversy behind its methodology and importance was about the first written record of influenza, as mentioned in the Bible.  While I'm not really one to judge the importance of others' research (what have I published in the last two years?  Nothing?  Oh, okay...), I can see why many would find this "research" distracting from truly important research.  Further, since this is a journal pertinent to my field, I can also see why some would think that a paper like this would lower the general impression of the journal.  I would feel slightly uneasy publishing in this journal knowing the qualify of the articles it has published in the past.

Still, I see articles similar to this Biblical influenza paper all the time.  And it makes me think that maybe there is some universal publication joke that I'm just not getting.  Are these papers published to slant the academic community toward some small fragment of comedy?  I doubt it.  Based on the titles, their affiliated institutions, and their content, I would say that these papers are just fluff, scientific nothingness packaged into a pretty little paper.  But why are the journals risking their reputations to publish this fluff?

And these are the papers that several hundreds (and thousands!) of people read precisely because they're funny, or ridiculous, or just plain stupid (maybe this is why the journals publish these papers!).  Now, I ask, who's the stupid one?  The authors who created and published the "ridiculous" studies, the reviewers that allowed the research to pass, or the masses of people that spent too much time scoffing and belittling the research?  Or is it me, the one who spent too much time composing this post about the papers and the readers?

Yeah, I think that I just answered my own question there.

The joy of summer recesses

A new emotion came over me as I drove into the parking lot this morning.  The parking lot was mostly empty, and I realized that it would remain like this for three more months.  Ahh, summer vacation for the medical students.  No longer will they be hogging the lot and parking as if they had just learned to drive.  Summer has arrived, and I love absolutely every second of it at school.

Not only is the parking arrangement so much easier, I also have no classes during the summer.  Essentially, I don't have to waste four hours of my life each week listening to lectures.  Instead, I get to perform cool experiments (and more of them!) and explore my creativity, which has admittedly wained during this last semester. Also, I can finally pick up that "extra" project I've had in my back pocket for the last year.  That one that I'm obligated to finish since we received a bunch of money for it.  Oof.  Yeah, I guess I'm glad I have so much extra time...

Even better:  fewer lab meetings.  My lab has a ridiculous number of lab meetings per week (three, to be exact) that really eat up time in the middle of the day.  Summer vacation means fewer lab meetings - now only one! - and, on top of that, there are fewer seminars and invited speakers.

Best: the weather is fantastic.  I'm looking forward to the days of sitting by the lake, reading a book, and just enjoying the summer heat.  Walking out of work when the sun is still shining is an added bonus.  Winter really can get my mood down, especially when I don't see the sun at all (no windows in the lab) during the day.  So it's finally nice to have a bright, blue, wonderful sky to greet me at the end of the day.  It's an added bonus that I can have my sunroof down as I drive into work and enjoy the cool morning air.

I've recently begun running outside after a lot of treadmill running.  Since I've never run in the summer, I'm not sure how I'll be able to handle it, but I think that some careful experimentation with when and how long I run will allow me to not collapse from heat exhaustion.  That and lots of water...

Zoom, zoom, zooming away

It's vacation time, meaning that I'm going to be away for a while.  No lab, no papers, no reading, no pipeting...  How exciting is that?  Plus I get to soak up the sun, drink lots of wine and beer, and do some awesome shopping (I hope).  Doesn't get much better than that...

The one downside to my vacation is that the timing is horrendous.  My project in lab has stalled because I can't clone anything, but just now my cloning is starting to work.  In an ideal world, I would get all of it done before leaving, but that's not quite possible.  Sigh.  The second bad thing is that one week after returning from vacation, I have a department-wide presentation to give.  While Super PI doesn't seem to think that it's very important, it is important to me, so I need to work on it a lot before leaving so that I don't look like a complete fool upon my return.

Anyway, I'm excited to be gone and looking forward to a lovely vacation.  İAdiós!

Organizing journal articles: Why must this be so painful?

I have a confession: I hate reading scientific papers.

And that's generally a problem because I'm a scientist, and the primary means of communication in the scientific world is via publications.  Still, I can't help but hate to read them.  Why?

Because they can be so incredibly dry.  Sometimes, I can really get into papers, and I can really enjoy reading them.  But the majority of the time, I just have difficulty keeping my interest (I blame Facebook).  What's even more problematic is that I cannot always remember what was done in papers that I absolutely must remember so that my PI doesn't think I'm a complete idiot.

I'm not sure if it's just me, but I just find it difficult to read papers sometimes.  Don't get me wrong, I can certainly read a paper, comprehend every word, and discuss the paper in its entirety to anyone.  I'm not stupid.  At the same time, I just find it difficult to spend an afternoon reading papers.  Maybe there are too many distractions, or maybe I just need to smack myself in the head.  But what is wrong with me?

I think that my main problem is organization.  I accumulate papers but I don't really read them thoroughly.  The folder on my computer of papers is several gigabytes, organized by first author, year, and article title.  But ask me to find a particular paper, and I'll probably just go to PubMed.  Then what's the point of saving and "organizing" all of these papers?  I'm still figuring that one out...

When I'm about to give a presentation or talk about science with someone, I generally mop up my act by reading several papers, looking at figures, and writing down some notes.  My problem remains that I cannot seem to maintain the knowledge, and that's a problem.

In the past, I've tried a few different strategies:

1) Writing small reviews of each paper I read.  I'll make a word document for every paper I read, summarizing the main findings and the figures, as well as my "verdict" (whether the paper is good or bad).  This i helpful to some degree, as it helps me both in going through all papers very thoroughly and making a judgment on the content of the papers.  On the other hand, it takes a lot of time and isn't particularly productive - I still forget a lot.

2) Just reading a lot.  The problem I've found with this is that reading a lot of papers is just overwhelming (sometimes).  I guess that I've overcome this by reading review articles and using that as a jumpoff-point for reading other articles.  That has seemed to be rather successful.

3) Organize papers into categories.  I've tried this several times, and it's never successful.  No matter how many folders I make or how carefully I catalog my papers, I can never find the exact one I want and eventually end up just going to PubMed - what's the point in that?  I've also tried programs like Mendeley, which I found to be frustrating.  Mendeley is a bloated program that takes to long to load, isn't fast enough, and doesn't have enough options for me.  I found it to be counter-productive.  I have yet to find a worthwhile paper-organizing software program.  Papers looks good, but I use Windows...

4) Print papers and make notes on them.  It seems that every professor has a cabinet full of old papers that they have scribbled on for their future use.  In my personal experience, I don't find having physical copies of papers to be helpful - it's more of a waste of space.  Plus, it wastes trees, and who needs that?  Electronic copies are also frustrating because it's more difficult to read on a screen.  Still, I find it better than having a gigantic pile of papers.

Is there something better I should do?  The answer is definitely.  I need to find a solution that works for me. It's wasteful for me to hobble along as I have been - and it's also kind of ridiculous that I've gone this far in my scientific career without figuring this out.  My newest idea is to read papers and draw little interpretations.  I love working in Illustrator, and maybe drawing out the main points of papers will make it more memorable - plus, it's never a bad thing to have these little graphics at my disposal!  We'll have to see how this little experiment goes...

A Necessary Evil: The Office Coffee

Is that warm coffee or a
steaming pile of crap?

It's one of the most deceiving entities in the department.  Sure, it may look friendly and like it might put you in a better mood, but, in reality, it is truly sinister.  After the first encounter, it may take a while to show its evil side, and it will likely have you coming back for more, time after time.

The coffee in this department is horrendous.  And I'm sure this isn't just a phenomenon of this department either.  On more than one occasion, I have had serious stomach issues after drinking the brown concoction, and you would think that this would steer me from ever drinking it again. But does it?

No.  Nearly every morning, you can find me filling my mug with the crap.  I try to improve it by adding some creamer and sugar, but I've found that this is useless based on the following careful testing:

One creamer package:  No change in flavor
Two creamer packages: Slight change in flavor, but not appreciably better
One packet of sugar: No change in flavor
Two packets of sugar: Oh dead god, what the hell just happened to my coffee?!
One creamer and one sugar: Slight improvement (synergistic effect, I suppose)
Two creamers and two sugars:  Modest improvement; too sweet and barely recognizable coffee flavor

I've basically found no solution to making that coffee bearable, but it doesn't stop me from drinking it almost every day. Sure, there's a coffee shop upstairs, but that costs money and, as a graduate student, I have too little money to justify spending it on coffee.

The best solution I've found is to buy flavored creamer and attempt to mask the flavor with something like hazelnut or Italian sweet cream (I don't know what it is, but I don't care because it is delicious).  The problem with this solution is that I use way too much of the flavored creamer to make that coffee even recognizable anymore.  Plus, it's got a ton of sugar.

My newest solution: black coffee.  If it's so bad, I shouldn't drink it, so maybe (maybe) if the coffee disgusts me morning after morning, I will wean myself from it and drink something better, like water.  The caffeine kick sure helps, especially during those long lectures, meetings, and presentations, so I'm afraid that I may just have to learn to live with the coffee.  Plus, it's free - I'm sure not going to complain about that.

The Slump

You know that all-to-familiar time during your research when absolutely nothing will work?  I've been going through this for about the last month, and it has worn on me extensively.  It has gotten to the point where even a simple plasmid mini prep won't work.  I'll check my antibiotics, use fresh media, make brand new compentent cells, but when it comes to the simplest of all, I cannot perform a mini prep.  I've determined that it is not a problem with the kit my lab uses because I'm not able to isolate any plasmids even using the "old school" methods of phenol-chloroform extraction.

In the past, I've gone through periods like this when I'll have a bad week, but it's never been this bad before.  Every time it happens, it makes me wonder how so much could go wrong.  After all, it is science, and we're supposed to have a method to our madness (emphasis on supposed to).  How can it be that a simple process that I was able to perform only a couple months ago has completely escaped me now?  Especially at a time when I need it to work the most...

Through my (admittedly limited) experience, I've found that there are only a few ways to drag myself from the pit of desperation:

1) Start several experiments and hope that one of them works.  This is my method of choice, mostly because I like to multi-task in the lab.  The important thing for me to remember is to make sure that none of these experiments are high-priority (I don't need these results right now with this experiment) and that at least one of them will work.  It never hurts to have a confidence booster, in my opinion.

2) Stop. While I find it hard to do, sometimes it just helps to stop and do nothing for a short period of time.  Usually this makes me feel even less productive and more depressed, leading to a vicious cycle.  I've found it can be beneficial sometimes, though, to just take a coffee break and stop worrying.

3) Read. Diving into the literature can never hurt, and reading can really take your mind off the sucky things that are going on in the lab.  I like to read reviews particularly when I'm stuck experimentally because it's less intense and allows me to relax a tad.  Reading more hardcore papers can also make you feel like your problems in the lab are quite insignificant compared to the 736 Western blots required for that awesome Nature paper.

4) Start over.  Like really start over.  New media, new cells, new antibiotics, new reagents, new pipet tips, new underwear, new music.  I find it's easier for me to pick up mistakes when I have to start over from the beginning.

5) Swallow your pride and ask for help.  Sure, a mini prep is a ridiculously simple task that a trained monkey (probably even a zebrafish) could do with one hand.  Still, it never hurts to ask for help and advice because there are probably people across the hall that have run into the same problem.  Even if not, it's nice to complain to others as a catharsis.

And that's where I'm stuck.  A month later and my mini preps still won't work.  I've tried all of my tips from above, and nothing seems to work.  All seems like it should be just dandy with a *%$! mini prep, but, alas, it is not.  As with all other times in the lab, I will plod along until one day, my ability to isolate plasmid DNA will magically reappear.  Until then, I will continue to complain about it... because that's what I do.

Post-slump update: My minipreps are all in working order again after I finally figured out that the media was an old formulation and didn't support plasmid replication for some reason unknown to me.  Lesson learned, I guess.  There is still hope for me!

An Introduction

Yes, yet another blog written by a graduate student.  When I created this, I knew that it would exist among the swath of other similar blogs.  At the same time, I consider it a fun outlet and opportunity for me to be creative, regardless of whether others read my posts or roll their eyes.

I'm a second-year graduate student, studying viruses and loving every second of it.  I've survived the first year curriculum, the qualifying exam, and the tribulations of absolutely nothing working in the lab.  I have enough experience in science to (usually) know what I'm doing at this stage, but I'm learning and maturing each day.  My dream is to be a successful principal investigator, and while the journey might be tough, I'm going to continue working my hardest to achieve that goal.

There is also more to me than just science:  I love traveling, graphic design, technology, eating good food, running and exercising, and reading.  I'm also the guy in the department that you will hear blasting the Spice Girls (every Friday), Britney Spears (mostly Saturday mornings), and Paula Abdul (Tuesday afternoons).  I sing along to the radio sometimes, I wear funky glasses, and I have an adorable car.  Overall, I like to think I'm quirky... a quirky virologist.