Thursday, November 11, 2010

It's been a while!!!!

So it has been quite a while since anyone has posted on our lovely blog and I refuse to let it die SOOO when a friend posted this on my facebook wall, I thought, what better place to share this than on our lab blog?!? LOLZZZ!!! Even though the definition sounds more like meowtosis.

I also thought that I would share the wonderful discovery that I made; Do you ever have SO MANY ARTICLES to read??? of course you do. Do you ever have SO MANY PLATES to dissect??? Of course you do! Did you know that in Adobe Reader, you have the option of having your article read out loud to you? Well you do! all you have to do is go to 'view', 'read out loud', 'activate read out loud' and you can have it read the page or whole article to you. You can even change the pitch and speed of the voice. It's better than listening to music. It will also kindly try to read the websites, sources and even sometimes figures in the papers to be read out loud. My point being that it's not great but it can be helpful so I thought I would share.

Lab Love,

Wednesday, August 4, 2010

Today I went hunting for Falcon Tube

and found this instead...

The various constituents of Olson lab were nowhere to be found. I suspect they are inside this machine.

Tuesday, August 3, 2010

Fun Fact of the Evening

Or morning, as the case may be.

I was reading about different types of blotting and the article (okay I admit it, it was the Wikipedia entry for Eastern blotting) had a fun timeline of all the witty scientists who have invented new types of blotting and carried on the tradition/joke of cardinal-direction-related names. (Southern blotting came first, and then Western, Northern, Southern, and a whole slew of sub-directions followed.)

I got very excited when I saw this one:

"(1984) Middle Eastern blotting has been described as a blot of polyA RNA (resolved by agarose) which is then immobilized. The immobilized RNA is then probed using DNA."

MIDDLE EASTERN BLOTTING!!!!! Hailing from the Middle East myself, this pleased me immensely.

From Sarah Moustafa on Picasa
The Middle East, minus the blotting.

And the paper that presented Middle Eastern blotting (mad props!), courtesy of PubMed:

Monday, August 2, 2010

A Closer Look: TCA Protein Preps

Last week, Amy (our sensational PI) and Esther (who, along with Tati and Rozi, has now absconded for home, leaving me to fend for myself in Mac Lab) came to me with a quandary-slash-question and I was pleased when I realized that, with the help of some chemical structures and pKa values, I could answer it! Amy was super excited at the structures that I drew on our whiteboard and suggested of her OWN volition (FOR ONCE) that I blog about it! So here, for you now, is my own understanding of how TCA protein preps work on the chemical level.

Some background. Esther has spent the summer working with proteins (whereas I have been focusing on genetics and cytology, and Rozi and Tati's project involved a genetic screen) - specifically, she was investigating whether proteins involved in regulation of the assembly of the synaptonemal complex (SC) in meiotic chromosomes are themselves regulated by posttranslational modifications that are dependent on recombination. Yes, that is a mouthful - to rephrase it, what she would like to find out is Spo11, the major protein player in recombination that catalyzes double-stranded breaks in DNA to initiate recombination events, also has an effect on the regulation of SC-assembling proteins by having upstream control over if and when these proteins are modified (i.e. by SUMOylation), if at all, as the modification of these proteins may be dependent on the occurrence of recombination events. So for example, if the SPO11 gene was knocked out in a strain, would there be any effect on the physical state (covalent modifications) of SC-assembling proteins - and if so, would this suggest that a lack of recombination sites on the chromosomes led to the SC-assembling-protein posttranslational modification pathway not being activated?

To answer these questions she was using Western blotting and, EXCITINGLY, 2D gel electrophoresis to basically check out the size of different SC-related proteins - Fpr3, Zip2, Zip3, Zip4, and Rrd1 - in both SPO11+ and Δspo11 backgrounds to see if there was any difference in size of the SC proteins in the two different backgrounds. BUT OF COURSE, before she could run these proteins out on gels, she had to isolate them from cells of her strains. And how did she do that? BY TCA PROTEIN PRECIPITATION. This is where I come in.

The TCA protein prep protocol is a relatively straightforward one - it involves variations on the basic theme of pelleting yeast cells, vortexing the pellet with TCA and glass beads to lyse the cells and release the protein, and then washing with a basic resuspension solvent to force the total cell protein out of solution as a pellet. Cool, right? But what she wanted to know was HOW adding some TCA to cells actually precipitated ALL of the protein in the cell, with no regard to specificity or anything. She had read somewhere that "hydrophobic aggregation" was involved. So I looked up the structure and pKa of TCA, and a lightbulb TOTALLY went off.

TCA is trichloroacetic acid, a derivative of acetic acid:

(Yes, this post, like the zygote post, is going to be illustrated with my superlative Paint skills. At first I was using ChemSketch, a really awesome molecule-drawing freeware program from ADC/Labs that you can get here, but then I got tired of it because the molecules all looked so uniform. I think molecules should have PERSONALITY.)

In aqueous solution, TCA dissociates into its conjugate base plus hydrogen ions:

Note the pKa, 0.77. That is VERY LOW. As you will recall from biochem, orgo, or wherever you learned about the lovely world of dissociation reactions, pKa is a measure of the acidity of a molecule - more specifically, the equilibrium constant for the dissociation reaction, and a measure of a molecule's affinity for its acidic proton when in aqueous solution (water). Since pKa is a measure of the RATE of the dissociation, and in general, the rate, K, of a reaction is given by-

K = [reactants]/[products]

reactions with a very high K (and in turn a very low pK, because pK = 10^(-K)) have a very HIGH ratio of reactants to products in solution. In our case, this means that in solution, virtually all of the TCA has dissociated into protons and conjugate base, because the proton so readily dissociates from the hydroxyl group.

(Of course, we could take this down even another level and point out that TCA has such a low pKa because of the enormous electronegativity of the halogenated end of the molecule - chlorine is the second most electronegative atom out-electronegated only by fluorine, and THREE chlorines all on one end of the molecule STRONGLY polarizes the electrons in the molecule toward that end - this enormous pull for the electrons away from the hydroxyl group means that there is a very small net electron density on the hydroxyl group, not leaving much for the proton to "grip" onto so it pops off quite easily.)

But of course, I lied to you in that drawing above. Free H+ atoms NEVER exist in solution. They ALWAYS exist coordinated to water molecules by hydrogen bonds. So really, the above reaction looks like this:

Notice anything? Given the extremely low pKa of TCA, its rapid dissociation means that practically ALL of the water molecules in solution will be "occupied" by existing as hydronium (H3O+) ions!

Now let's zoom out to Esther's experiment. This is why I love biochem.

Esther's protocol called for adding TCA and glass beads to a cell suspension, vortexing vigorously, centrifuging and collecting the supernatant, and then pelleting out the protein from the super by resuspending in Tris base. NOW WE KNOW how the TCA makes the protein fall out of solution! Do you see it yet? (The glass beads and vortexing were simply physical trauma to the cells to lyse them and release the protein and other cell contents.)

When you add TCA to an aqueous solution, it dissociates IMMEDIATELY and the ENORMOUS concentration of protons intercalate with the water molecules, effectively blocking them from solubilizing the cellular protein (which is mostly hydrophilic, as most cellular proteins have at least one hydrophilic region). Since the TCA outcompetes the protein for hydrogen-bonding with water, the proteins have nothing to do but aggregate with themselves and fall out of solution as hydrophobic aggregations.

This picture is actually most accurate because each water molecule is hydrogen-bonding with two protons - because the oxygen in water has two lone pairs, each of which can coordinate one proton. Then when you spin this whole mixture at high RPMs, cell debris (like the cell wall and membrane and organelles) and protein fall completely out of solution, while water keeps the TCA busy (or vice versa, as the case may be) in the aqueous layer. Tossing out the aqueous layer yields a pellet containing all of your cellular protein! The pellet also contains unwanted cell debris, so a wash and spin in a very basic resuspension solution (i.e. Tris base) again forces the cell debris (which are not that polar) out of solution, FINALLY yielding a solution of all the cellular proteins.

Really cool chemistry behind a useful, (seemingly) straightforward protocol. :)

Sunday, August 1, 2010

Five Ways Not to Grow Insect Cells

I'm kind of afraid Sarah will sneak into my room at night and shave my eyebrows off if I don't here it goes.

I (along with most of the other contributors to this wonderful blog) have recently successfully completed the Hughes Program. After working in our labs every day, all day for ten weeks we're finally done with our projects. Or at least that was the idea. I am nowhere near done with my project.

Swastik, my lab's awesome grad student (I, too, am not a part of the MacQueen lab), told me the other day that when Thomas Edison finally succeeded in making the light bulb after his 30th attempt (or something like that), someone asked him if he felt like he was wasting his time for all those years he spent unsuccessfully making the light bulb. Apparently, the wise Mr. Edison replied that he did not, in fact, feel as though he had wasted his time because he now knew of 29 ways not to make a light bulb.

Well, I've spent the last 10 weeks growing insect cells, and I've learned at least 5 ways not to grow insect cells. I'm using insect cells as a way to express a eukaryotic protein and make a lot of it in hopes of one day crystallizing it and finding its structure. The cells I'm using are ovarian cells from this caterpillar, called the cabbage loper (Trichoplusia ni):

Because they're just the ovarian cells, they don't have any kind of immune system, so they have to be cultured with sterile technique. This means I have to work with them inside a sterile hood, spray everything that goes into the hood (including my hands and arms) with ethanol and generally make sure I don't contaminate them. If they die, then I have to start over from a frozen stock.
In order to make them express my protein, I infect them with a virus called baculovirus. In the wild, I'm told this virus causes the caterpillars to burst and drip down onto the caterpillars on tree branches below them, infecting these caterpillars as well. In the lab, we use a virus that has the instructions for making our protein in it, so before it kills the cells, it causes them to make a bunch of protein.
Anyway, I'm the first student in my lab (the Olson lab) to use this expression system, as we, like the MacQueen lab, just started last fall. Therefore, it's been kind of an adventure for me to figure out what to do and not to do, and while I did manage to express enough protein to squeeze out some data this summer, I also discovered at least five ways NOT to grow insect cells. Because I did not have a chance to share this accomplishment on my Hughes poster, I will take the opportunity to do so here.

Five Ways Not to Grow Insect Cells by Falcon Tube:
Don't grow insect cells by...
1) ...Letting them get too hot. This is tricky if you're working somewhere where they keep turing off the AC when it gets above 95 outside (as they do at Wesleyan).
2) ...Being too obsessed and look at them every day twice a day, as you'll shake them around too much and they'll die, or at least be unhappy and grow slowly.
3) ...Growing them without antibiotics. They can get infected with bacteria and die. (Some people do actually grow them without antibiotics, but it didn't work for me).
4) ...Letting them get infected with fungus. They will die. (There's nothing you can really do about this except being extra sterile).
5) ...Letting all of your cells get infected with your virus. All of them will make your protein...but then they'll die. This makes it a) impossible to have negative controls b) really hard to get anything done.

So now I know all of these things can happen to insect cells. And I know (at least a little bit better) how to stop them from happening. I'm proud to say I've had happy, living insect cells for about two or three weeks now, and I hope to keep them that way for a productive semester.

Photo credit:

Tuesday, July 27, 2010

Now We Can Be Bffls!

I was told by Sarah that if I did not write on the blog of the esteemed MacQueen lab, we could not be best friends. Therefore, I must post!

A few members of the MacQueen lab tentatively came into the Hingorani lab the other day to look at my microplates. In the microplates were fabulous colors (whose beauty was magnified by the photography skills of the MacQueen gang--see 'Sourjourn into Manjuland").

I am working on characterizing the kinetics of three MutS (Msh2-Msh6 in humans) mutants. MutS is a protein that recognizes mismatched DNA and recruits other proteins to initiate the DNA mismatch repair process. I am studying three cancer-associated mutants in Thermus aquaticus ("Taq"), as it is an easy organism to work with and there is high sequence homology between Taq and human MutS. In addition to binding mismatched DNA, MutS it is also an ATPase, and we're interested in the mechanism of ATP hydrolysis (ATP--> ADP + Pi) in the MutS mutants, and it is thought that MutS may communicate that it has bound a mismatch through coordination of its DNA binding site and ATPase domain.

One way to measure ATP hydrolysis is through phosphate production. The microplates that the MacQueeners photographed were part of the "Malachite Green Assay." Malachite green turns from yellow to green when it binds phosphate and this color change can be quantified. We create a standard curve with our stock of phosphate and then use this standard to calculate the concentration of phosphate at different time points in the reaction mixture that includes MutS and ATP. Using the concentration of phosphate at different time points in the reaction, we are able to determine kcat, which informs us about how fast the protein hydrolyzes ATP (more formally, kcat represents how many molecules of ATP are hydrolyzed per MutS active site per minute).

So there we go. Those are the pretty colors.

Sarah, now can we be best friends?

Saturday, July 24, 2010

On WEAST- Edit

So I would like to edit my previous post. I neglected to mention some important information; the other labs that present at the WEAST meeting are the wonderful Holmes lab, with whom we went on a hike and you will hopefully see pictures soon, and the fabulous McAlear lab. The other person presentin today (bc more than one person usually presents at WEAST) was Sarah Kass-Gergi, a brilliant undergrad from the McAlear lab. Hopefully we can convince her to write a post sometime in the future.

Sojourns in Manjuland

Today, something very special happened.

We got to visit Manju's lab.

Let me give some background information so as to explain wh
y THIS IS A HUGE DEAL. Dr. Manju Hingorani runs the lab next door, and I think she is kind of enigmatic, and very awesome. She keeps a little tiny tote bag stocked with chocolate hanging on the doorknob of her office, and she's always pacing around with a canteen of loose tea leaves and whispering about DNA. I'm not so sure the admiration is mu
tual, though, because in the past, Manju (who, by the way, INSISTS that everybody, students included, refer to her as Manju and gave me a death look the first [and last] time I referred to her as Dr. Hingorani) has expressed her, um, dissatisfaction with some of the more robustly-vocal-chorded undergrads of the MacQueen Lab
(one is the author of this post and the other has a nickname that rhymes with "gators")
and their propensity for emitting cackles that reverberate into her office. Given this stormy torrid drama of our past, I always thought setting foot into DNALab would be a mere dream, never to be fulfilled...

BUT NAY!!!!!!!!!!!!!!!!!!

Today after WEAST (which will be blogged about in the near future for those of you not in the know), Tatiana came galloping into our lab proclaiming "ARIEL IS DOING THINGS WITH PRETTY 96-WELL PLATES!!!!!!!!!!!!" Ariel is the awesome undergrad in Manju's lab who, as Esther informed us, placed first in her group in the Middletown 5k!! Anyway, I grabbed my camera and scurried to investigate.

HOW COOL IS THAT?!?!??!?!?!?!?!

More science eye candy:

I know at this point you're probably thinking "Wow, that IS pretty, but what IS it?" (either that or "This girl needs to get out more"), and I am embarrassed to say that in my haste to get back to work (okay, it was actually because Ariel made us leave), I didn't ask her what exactly these were for. But I PROMISE I will find out and update next week because I know you're all as curious as I am!!!!!!!

P.S.: Get excited for a SUPER-SCIENTIFIC (seriously!) post tomorrow about the biochemistry behind SPORULATION! It's one of the most useful and important techniques that we use in our lab, but admit it, do you really know why putting yeast into media containing potassium acetate makes them enter meiosis (beyond "it starves them")? FIND OUT HERE TOMORROW!! (I was actually going to write this post tonight, but then I ended up spending more than an hour JUST ON THE RESEARCH. This is legit, folks!)


Hey y'all! I'm writing a breif entry about our WEAST meeting. Since everyone has been scolding me for not having blogged yet, I figured better a short entry than no entry at all.

Today we had our last WEAST meeting of the summer :(. As sarah would say, sad day. WEAST meetings are these wonderful meetings that we've organized with some of the other labs that use yeast ( hence weast, wes yeast). They are super helpful for learning how to present your material to others, dealing with questions and troubleshooting. I think it is very nice to have meetings with people who know the organism and can bring different perspectives and advice to the table on a particular project.

Since today was our last day of the summer we had tons of food (nom nom). Rozina and I presented the work that we have been doing, which is basically to create a tool, in our case a temperature sensative allele, that will allow us to investigate the role of Zip2, Zip3 and Zip4 in synaptonemal complex, the protein that holds homologs together during Meiosis I. In case you were wondering what the above picture is, it was a drawing I did for our presentation of abnormal and normal synaptonemal complex.
I think the presentation went more or less smoothly, there were a couple of questions that I had some difficulty with but overall it was a great learning experience. We have presented our work a few times now and each time we learn something new about how to present, or the content itself. I hope that WEAST will be able to continue into the year and I think that it's the kind of meeting that all labs should be doing in their field on a regular basis because it promotes sharing of information, ideas and thereby progress and forward momentum

Friday, July 23, 2010

Things that need to be addressed...

Nicki Minaj > Lady Gaga
(greater than)

weekend project?

Sarah, are you a giant puddle of red goo yet?


seeing as to that I'm on "vacation" I'm going to troll this blog n not put science related things up, until next week of course

Thursday, July 22, 2010

5k citizens FUN run!!

Yay my first post!!

Yesterday Amy and I ran a 5k race in middletown with some other MB&B students and faculty so Sarah asked me to blog about it.

It looked like it was going to rain during the race but miraculously, the storm stopped just for the race. The weather was perfect for the race. The race was about three miles long and it looped around william street/college street and around washington terrace/usdan. After the race we all met up to take a picture and to eat pizza, bagels and cookies.


The Wesleyan MB&B team also place fifth overall :) yay for us!!!
Overall the race was really fun and I hope that more people from our lab will participate next time

goodbye for now

Shmoo you.

As everybody who works within a 50-yard radius of our lab knows, I love zygotes. THEY ARE SO CUTE. Picking zygotes is one of my favorite things to do in lab, mostly because they are SO CUTE, but also because pure diploid colonies - i.e. those that grow up from zygotes - sporulate MUCH better than mixed diploid/haploid populations - where not everybody that hits that spor plate can sporulate in the first place because HAPLOIDS DON'T DO MEIOSIS!!! I sporulated some particularly robust zygotes a few days ago and the tetrads were BANGIN'. It was delightful.

So anyway, I wanted to find a nice picture of a zygote on Google Images to post here so that you all could coo over zygotes with me, but I found Google's results unsatisfactory, so I decided to draw one myself.

Shut up.

Everybody loves science.....even Public Safety

Alright, so after much poking and prodding by Sarah and Tatiana…and then reading something about tears (see entry below), I have decided to make my first contribution to the blog. Bear with me.

So last week, while sitting at my job in the Science Library and doing my daily tasks (twiddling my thumbs, watching an episode of Felicity, spinning in the chair, etc.), a bored Public Safety officer strolled up to the counter and stared at me until I finally agreed to look up and make conversation. After talking about random things like the weather, Lebron James, Pattie Palace, and how he was a court martial (and not an employee at Marshalls like I originally thought he said), we somehow came upon the topic of how I was a Bio major that was working in a lab this summer. Upon hearing that, the excitement could not have been more evident on his face.

Excited Public Safety Officer: Do you guys work with mice?

Me: No, we work with yeast.

Until I said that of course.

Even so, he continued to go on and on about how he watched something on television that showed scientists growing a human ear on a mouse, and how crazy and exciting the idea was, and how amazed he was, and how he never thought such a thing was possible….and so on and so

forth. And of course, being in the field that I am, I could not help but not only feel the same excitement as the officer, but also the urge to learn more about what he was talking about (see here:

But in addition to this, the conversation with the officer had me thinking. I could care less about math or history. I couldn’t initiate a conversation with anyone on a topic of interest in economics, philosophy, or religion. But everyone…yes everyone, loves at least some aspect of science.

Even if it’s just Bill Nye the Science Guy, because everyone loves him too.

Good night.

I'm going to replica plate my TEARS now!!!!!!!!!!!

Ok guys, none of my supposedly "AWESOME" "FANTASTIC" "OMG SARAH I HEAR YOU CACKLING THROUGH THE WALL LET'S BE DINO BIFFLES IN OUR BLOG FOREVS TOTES" labmates/fellow bloggers have found time in their BUSY SCHEDULES to write anything new and inspiring (Taterz, I'm glaring angrily at our shared wall, nay, FROWNING), so I'll just have to take matters into my own hands.

THAT BEING SAID. Did you know that when we create a yeast strain of interest (i.e. a precious haploid of the correct genotype and mating type obtained from approximately 1.5 billion hours of dissection), we save it for future use (i.e. for ourselves or to share with labs who might want to work with said genotype) by literally FREEZING it - sticking it in that
huge -80 degree celsius deep freezer in the alpha room (aka Esther's cave), and the yeast are totes fine when we go to get them out a month or a year or 30 years later? I THINK THAT IS SO COOL. All you have to do is get some:

-30% glycerol (about a ml per strain you want to freeze)
-Cryotubes and tough-labels (the round white ones)
-Deep freezer (-80 degrees C)
-Lab strain book
-Oh, yeast strain(s) too. Preferably useful ones.

Put a ml of glycerol in the cryotube. Glycerol is a viscous fatty acid that protects the cells from the harsh freeze-thaw cycles they endure - that's why we freeze in glycerol instead of plain water.

MY BFF GLYCEROL!!!!!!!!!!!!!!!!!!!!!!!!!!!!

Then just place a clump of cells in the glycerol, shake it like a Polaroid picture to resuspend them, and freeze them! Then write the full genotype of the strain along with the freezer number in the strain book.

Ok that part was boring but REALLY THE COOLEST THING ABOUT ALL THIS (people who are not interested in reading protocols can start reading here) is that when you go to get them out of the freezer you don't even have to wait for the tube to thaw to remove some of the liquid!!

Karen: You can just scrape a toothpick across the top of the frozen glycerol and streak it onto your YPD plate. They'll grow right up.
Sarah: OHEMGEE, that is like SO COOL!!!!!!!! OMG, YEAST POPSICLE!!!!!!!!!! Can I try it RIGHT NOW?!?!?!?!?!??!
Karen: No.

Sad day.

Wednesday, July 21, 2010

WHAT UP CELL CULTURE!!!!!!!!!!!!!!!!!!!!!!!!!

Greetings from Fuzzy (and Jr), our lab contamination pet.

We are part of the MacQueen lab at Wesleyan University and we would like to welcome you to our lab blog, called "Cell Culture"! In case you haven't noticed, we are super nerdy and think that most everything science is fun. So don't be fooled by the title, this blog will be about the serious (and also the not so serious) aspects of cellular biology and the culture of the cell bio community. And by community we mean mostly our lab.

With love from Cell Culture's creators,

Sarah and Taterz