Mimosa

If there's one thing my blogs lack, it's regularity. This blog, for instance, reads like the fossil record of the earth. Gaps appear and there's no way to guess what's happened in the intervening period. And I don't make any attempt to fill them in. It made me feel guilty at first, but now i'm used to it :)

Anyway, I'm back in college now. Vacation's almost over, which means it's time to get back to the lab. I'm working with a different group this term. This group works on plants. Mostly potato and tobacco, but they're also starting work on the touch-me-not plant. It's suspected that touch-me-not uses many of the same signal molecules as our nerves, which is very cool.

However, until we get to the studying what works how and where, there's a lot of groundwork to be done. For one thing, not many people work on the touch-me-not, so we need to come up with standard, repeatable ways to study everything we study. Otherwise there's no way anybody can verify what we find out. I'll keep you posted as things keep moving.

The weather in Pune is beautiful right now. The roads are wet and muddy, but that's another story. I can see hills outside my window, and they're all covered by a thick carpet of green grass.

It's a good time to be back.

And this is something I found online (sorry, physicists!)

Turning up the heat

I'm on vacation now, and the project reached a... well, not much of a conclusion really. I had to make a presentation right at the end about what I'd done over the last 4 months. And the gist of my presentation was, "It didn't work". You probably saw that coming, what with the cells dying and all

But hey, that's what life in science is about. A successful scientist is one who'd make King Bruce of Scotland look like a quitter of the first order. Nothing you learn in school really prepares you for life in the lab. Come to think of it, nothing you learn in school prepares you for life itself. But that's a topic to be discussed another day.

A couple of days back I was reading this article in The Hindu. And a lot of what it says makes sense. Any bug has to strike a balance between the way it spreads, and how virulent it is. An extremely virulent virus would kill its host before he/she could come into contact with another potential host. Dead host means the virus ain't going anywhere, and consequently the virus is wiped out.

But in a city where you have a few hundred thousand people per square km., it's not difficult to find another host. Especially when we make things easier by squeezing into buses and trains. That's why cities need, absolutely must have, efficient sewerage, water supply and food supply. (Right now, there's an outbreak of cholera in parts of Hyderabad, where I'm currently at. And that just makes the dangers of poor sanitation extremely clear.)

I remember reading about a study where they looked at typhoid outbreaks in South America. They found that the disease was less deadly in countries that had proper sanitation. Apparently this was because good sewerage systems made it difficult for the disease to spread. So it had to hang around in one host longer, to improve its odds of finding another host. And as I said, a bug in a dead host is eventually a dead bug. There was a selection pressure for less virulent bugs, and so less deadly strains evolved. (Score one for the Darwinians!)

The take away? Usual summer advice. Keep yourself hydrated, but try to stick to boiled water. And watch out for cut watermelon sitting in roadside stalls!

They call me the cell killer.

Summer is here, apparently. That’s what one of the PhD students told me. Why? The summer interns have arrived. A boy and a girl. Yes, already. They study in Pune, so they’re getting a head start even though their vacations haven’t started. Plus side? I’m not going to be at the bottom of the lab pecking order now! Both of them seem nice enough. And the girl isn’t hard on the eyes, so that helps :D

It turns out that you can kill Schneider cells (the fly cells) as well. At least it wasn’t a spontaneous mass suicide like those fussy HeLa cells I was working with last semester. Yes, I know for a human derived cell line, HeLa is pretty hardy. But you wouldn’t think so if you could see the kind of abuse S2 can take in its stride and keep going strong. I mean, these cells can survive for 15-20 days with no medium change, nothing. HeLa would’ve died 3 times over by that time.

Anyway, back to the dead S2 cells. It was human error this time. I put the cells in a petri plate in an incubator without a humidifier. Left it there on Friday. Monday morning, the plate’s dry. I mean, properly dry. Why didn’t anybody realize this could happen? We’ve been growing S2 in flasks till now. The flasks have screw-on caps, and S2 cells aren’t too fussy about oxygen concentration. So we screw the caps on tight, and the medium doesn’t evaporate.

The take home message? Petri plates with liquid medium need a humidifier. I’m learning, see?

West side story

About two months back, I’d promised a post about western blots. So here goes.

The principle behind a western blot is the same as electrophoresis. Proteins acquire a negative charge in an appropriate solution. So when a potential gradient is applied, protein molecules will naturally move to the positive electrode.

However, not all proteins are of the same size. So if we place some kind of size-based filter between the electrodes, the smaller molecules would move to the electrode faster. That is the function of the gel in gel electrophoresis. There are pores of a certain size in the gel (the size of the pores can be varied based on the ingredients used) So under a certain potential gradient, with a gel of a certain pore size a protein of certain size/mass can only travel a certain distance in a certain period of time. So a mixture of proteins can be separated on the basis of size by gel electrophoresis. First we stain the proteins before we add them to the gel. That way, we can see where the proteins have been added (A protein solution in water is usually colourless) Alongside the proteins that we study, we also need to add markers. Markers are proteins of a predefined size, which we use like a ruler to gauge the size of the protein we’re studying.

Now that we’ve separated out the mixture of proteins, we need to make sure that the protein we’re looking for is in there. That means detecting with antibodies, since antibodies are extremely specific with the proteins that they bind to. But antibody staining can’t be done in a gel. So the proteins need to be transferred to a durable film of nitrocellulose or polyvinylidene fluoride (PVDF). Once the transfer is complete, antibody staining is done. Staining is a two step process. First, we stain with an antibody that’s specific for the protein we’re interested in. Next we stain with another antibody that’s both specific to the first antibody, and also has fluorescence activity

This is important because it simplifies the procedure. To induce fluorescence activity in an antibody, it needs to be modified artificially. By separating protein recognition from fluorescence, we only need to produce one kind of secondary antibody, which will bind to all primary antibodies regardless of what protein the primary antibody binds to. Instead of building fluorescence into primary antibodies, which means tinkering with pretty much every antibody produced for western blotting.

Why is fluorescence important? It helps us see precisely where our protein is located on the PVDF film and, as a result, on the gel (since the film is like a photocopy of the separation pattern on the gel)

Although I’ve simplified things here, the whole procedure takes a day at least to carry out. Electrophoresis can take anywhere up to 4 hours depending on the protein you’re studying. Transfer of protein onto a PVDF film is done overnight. Primary staining takes three hours, secondary staining takes an hour. And after each staining, the film needs to be rinsed for an hour, otherwise there’s a lot of background fluorescence and you can’t see your protein of interest clearly.

In other news, I’ve shifted lab groups for this semester. My previous guide is on maternity leave till April, so work in her group will go slowly till then. This semester, I’ll still be working on cell cultures. But this time it’ll be fly cell lines, not human. These cells are generally hardier than both HeLa and 293T, so I should have an easier time, in terms of cells dying, over the next few months. Wish me luck!