Immunofluorescence Step 2, Part 2

Previously, on Attack of the Clones….

Mr. Protein O’Interest was standing next to the bathroom in Millenium Park (or the boathouse in Central Park, etc) wearing a distinctive hat. An A. Body clone had recognized the hat and grabbed onto it. A group of ten Clone Grabber (CG) clones had recognized various bits of apparel of the A. Body clone and grabbed onto them. And, you are high overhead in a helicopter and it is night-time and all is dark. You’ve made your target slightly bigger, but you still can’t see it.

Time to shed some light on the situation.

This procedure is called immunofluorescence for a reason. And that is because the secondary antibodies (the CG clones above) have a fluorescent molecule attached to them so that, after they are put onto my sample, I now have a large glowing dot in my cell. In the park analogy, this is somewhat equivalent to having each of the CG clones wear a miner’s cap with a lantern on it. Now, from your helicopter, you can see a glowing spot on the ground and that spot tells you where the bathroom is!

Actual fluorescence is a bit more complicated than that. In order for the molecule to fluoresce, you first need to shine light of a particular wavelength on the molecule which will then emit light of a different wavelength (for reasons of physics*). In my case, I am shining green light on the sample and the secondary antibody then glows red.

There are some differences between the park analogy and real-life immunofluorescence (you know, besides the facts that I’m looking at a cell, not a park, and proteins, not clones). First, I’m not looking just at one molecule of my protein. If I was, I probably wouldn’t be able to see it even with the signal amplification I get from using a secondary antibody and the fluorophore. There are many, many, many molecules of my favorite protein in the cell (ie Mr. O’Interest is a clone himself and all one hundred of them are surrounding bathroom). Additionally, my cells have been “fixed.” Before I ever even put antibody on my sample, I have incubated my sample in formaldehyde which kills the cell and freezes it in time. This is because in order to get the antibody into the cell to begin with I have to do some fairly harsh things to the cell that would kill it anyway. In the case of my yeast, I have to destroy the cell wall and then make the remaining plasma membrane permeable to my antibody solution (both of these things allow the antibody into the cell so that it can recognize my protein). These things make the cell very unhappy. The advantage to killing the cell and fixing it with formaldehyde is that it kills the cell very fast and preserves the intracellular environment as it was just before I killed it (it’s a bit like the volcano erupting in Pompeii and gas and lava killing everything so quickly and freezing the scene so well that they were actually able to find people in the midst of whatever they were doing when the eruption started) (I love analogies, in case you haven’t noticed). The disadvantage is that the fixation process causes some artifacts that need to be dealt with (ie the people of Pompeii were probably not uniformly gray nor did they spend their days huddled in a corner with their faces in their hands). Also, you can’t view a process when everything is frozen in time, you can only infer what was going on based on the picture you now have.

And, for years, scientists lived with these caveats and we were mostly happy to do so because it allowed us to get data that we wouldn’t be able to obtain otherwise. But, still, if you wanted to view intracellular dynamics, you were pretty much SOL.

But one day, people thought, “You know, if we can attach an epitope tag to our protein, couldn’t we attach something else to our protein? Something that fluoresces all by itself without the need for chemical treatment?” And that’s exactly what they did.

Next Technique Series: Direct fluorescence——————————-

*Physics is a subject that I am not particularly good at, but which my husband is very good at (he’s an astrophysicist). Therefore, I try to avoid explaining physics if at all possible, leaving it for him to handle.

Probably not the actual lyrics

You know how, sometimes, even though you know what you’re hearing cannot possibly be the real lyrics to the song, your brain really can’t figure out what they ought to be? Well, this is what my brain heard recently:

In my mind and in my car, we can’t rewind we’ve gone to far.
Pictures came and broke your heart, put the blame on PCR.

From “Video Killed the Radio Star” by The Buggles.

Something tells me my brain would not have come up with this had I not been listening to the song in lab.

Immunofluorescence Step 2, Part 1

So, when last we left our Mr. Protein O’Interest and the A. Body clones, the A. Body clones had each attached itself to the hat of a different Mr. O’Interest in a different park. As I mentioned before, the fact that the A. Body clones have attached themselves to the Messr. Interest hardly helps you to find the bathroom, boathouse, ranger station, or campground since it is well nigh impossible to see people from high up in the air.

This is the situation I am in with my sample of cells after step 1. I have put antibodies on my sample that will recognize the epitope tag on my protein and this in turn will tell me where the ER is in the cell. However, proteins are incredibly small. So small that you cannot see an individual protein even if you magnified it 1 million times (and I assure you, the microscope I’m using does not have that sort of magnification capabilities). The fact that I’m now looking at a complex containing two proteins doesn’t help me very much.

So, the next step in the procedure is to 1) give me something to amplify the signal–so that I’m not just looking at a complex of two proteins–and 2) to give me something that I can actually see. This is, again, done with antibodies. However, these antibodies are a little more indiscriminate. They will recognize anything that came from a mouse (remember, my first antibody–called the primary antibody–originally came from a mouse). And, these secondary antibodies will recognize a variety of parts of the first antibody. This means that you will have many molecules of the secondary antibody attached to one molecule of the primary antibody.

Back to the park analogy. Okay, you’re high up in a helicopter and below, an A. Body clone has found the hat-bearing Mr. O’Interest and has grabbed onto that hat. Now, you release a second set of clones into the park–the Clone Grabber clones. The CG clones are a mixed group of clones. They have been trained to recognize the A. Body clones that are in the park, but they have been trained to recognize different parts of the A. Body clones. Some of the CG clones recognize the A. Body shoes, some recognize the A. Body right pantleg, some recognize the Star Wars insignia on the A. Body t-shirt, some recognize the A. Body hair, etc., etc. Therefore, when the CG clones find the A. Body clones, each of them will grab onto the part that they recognize–the shoe-recognizing clones to the shoes, the pantleg-recognizing clones to the pantleg–such that in the end you have quite a number of CG clones attached to the A. Body clone (who is, in turn, holding onto Mr. O’Interest’s hat). So now, instead of having a group of two people to detect from your helicopter, you have a group of 10 people to see. But, if you’ve ever been in an airplane flying high over a city, you will know that is not enough. Especially if you are flying over it at night. When you are flying over it at night, in fact, mostly all you see are lights….

To be continued…..

Immunofluorescence

A huge portion of the experiments I’m doing involves a technique called immunofluorescence (IF). Therefore, I thought I’d spend some time (several posts, actually) explaining what that is.

IF uses antibodies in order to identify proteins inside the cell that you are studying causing them to fluoresce under particular wavelengths of light. This allows you to see where in the cell a particular protein is found (we would say, where the protein is localized within the cell). In my case, I am looking at proteins that are always found on/in a certain place in the cell (the Endoplasmic Reticulum (ER), actually) and therefore can be used to mark (or “label”) that particular region of the cell.

Antibodies are a type of protein found in the immune systems of higher organisms. An antibody recognizes a specific protein (or part of a protein) and binds tightly to it. In your immune system, this occurs so that your body can identify that protein as foreign (because it’s attached to a virus or bacteria) and therefore target it for destruction. Immunofluorescence takes advantage of the specificity of antibodies for a particular protein in order to identify a protein in the crowded environment of the cell.

How does this all work? Let’s say you are in a helicopter, high above the ground, and need to locate where a particular structure (let’s say the bathroom) is in Millenium Park in Chicago (aka the ER) on the evening of July 4 when everyone and their brother is in the park. You can’t really identify the bathroom from in the helicopter because it is the same gray as the concrete surrounding it. However, you know that Mr. Protein O’Interest (aka my protein) hangs around the bathroom so if you can find Mr. O’Interest, you have found the bathroom. Now Millenium Park is huge and there are millions of people there, milling about (this is true in a cell, too; the volume of the cell is huge with respect to the size of a single protein and there are millions of proteins inside the cell). How do you locate Mr. O’Interest? Well, if you train a large number of A. Body clones (the antibody, of course) to recognize the face of Mr. O’Interest (which is presumably different enough from every other face in the park and therefore will specifically identify him), you can send the clones out into the crowd and when one of them finds Mr. O’Interest, it latches onto him.

This process is the basis of immunofluorescence. I want to find the ER in the cell. To do that, I need to locate a protein that is always at the ER. To find that protein, I dump some antibodies on my sample. The antibodies are specific for my protein and when they find it, they latch onto it (so to speak).

So, how do you get antibodies for your favorite proteins? Well, you expose an animal (often a rabbit, but in my case, the antibody comes from a mouse) to the protein by injecting purified protein into them. The animal’s immune system then creates antibodies to that protein (like how you trained the A. Body clones to recognize Mr. O’Interest) and the antibodies are collected and purified.

Generally, a researcher will make purified protein and then send it off to a company that specializes in making antibodies, who will then send the antibody back to you. This can be a very expensive process, though, and it can go wrong in any number of ways leaving you out a whole lotta money with nothing to show for it. So, what’s a researcher to do?

Fortunately, there are companies who mass produce antibodies for commonly studied proteins. However, these antibodies are usually for mammalian versions of the proteins and almost never recognize yeast versions. So, researchers have come up with a way around this problem.

Remember, I said that antibodies can recognize a particular region of the protein. That region can be really quite small. So, somebody figured out that if you took the antibody recognition region of a specific protein (let’s call it myc) that already had an antibody made for it, then stuck it on the end of your favorite protein.* The antibody would stick to myc which was in turn attached to your protein, effectively giving you an antibody to your protein. Which means that you don’t need a different antibody for different proteins. All you need to do is attach myc to your protein and use the myc antibody. These protein regions are called epitope tags and there are a few commonly used tags. Because so many people use these tags, companies have mass produced antibodies to those tags, thus saving the researcher from having to have a custom antibody made. Very nearly all of the IF I do uses antibodies for epitope tags.

Let’s put this into the context of the Millenium Park example. Let’s assume that you do not just want to find the bathroom in Millenium Park, but also the boathouse in Central Park, the ranger station in Yellowstone Park and the camping grounds in Yosemite Park. Each of these locations has a different Mr. Protein O’Interest hanging around. Now, you can train separate, customized groups of A. Body clones, each recognizing the face of a different Mr. O’Interest (very expensive and time consuming), or you can have each Mr. O’Interest wear the exact same kind of hat (the epitope tag)**–a hat that nobody else in the park would wear–and purchase a single group of hat-recognizing A. Body clones from a company. The company has put the time and energy into training the A. Body clones into recognizing the hat. All you have to do is buy the clones and set them loose on the various parks to locate the hat-wearing Mr. O’Interest and therefore, your structures (bathroom, boathouse, ranger station, campground) of interest.

Now, you are probably wondering how it helps to have an A. Body clone attached to Mr. O’Interest because all we’ve really done is replaced recognizing Mr. O’Interest with recognizing the A. Body clone. For that matter, how is it easier to see Mr. O’Interest in the first place? Isn’t the bathroom bigger than Mr. O’Interest? Wouldn’t a larger object (even if it blends into the surroundings) be easier to see from a helicopter than a much smaller one? This brings us to step 2 of the immunofluorescence process.

To be continued……

—————————-

*This of course raises the question of how one sticks a bit of one protein onto another protein. The answer is that you put the DNA coding for the tag (bit of protein) into the DNA of the protein you want to attach the tag to. Which raises the question, how do you put DNA from one protein into DNA from another protein? Well, it’s complicated. I’ll have to have another series of posts just for that.

**How Mr. Interest gets the hat will be dealt with another time (see note on about tagging a protein).

Lab update 2-15-08

In Lab: 

This week has been frustrating in several ways with regard to labwork.  First of all, I am attempting to optimize a particular procedure.  “Optimize” is code for “doing lots of experiments that are ultimately failures.”  While, in truth, you are actually moving forward because the optimization needs to be done and each parameter you tweak gets you closer to your goal of the (almost) perfect experiment (there is no such thing as the absolutely perfect experiment), it actually feels as though you are getting nowhere fast and have nothing to show for the 60 hours you just spent in lab.

The other reason this week was frustrating was because I ran smack into my own ignorance.  We have this image processing software that we use for the images that we take with the camera on the microscope.  I had been just merrily using the software, following the steps written up by someone else, when I ran into a problem.  When you run into a problem, that’s when you find out how well you know the system.  The answer for me was, “Not very well at all.”

The process I wanted to do is called deconvolution.  Before this week, I knew that deconvolution “made your image look better.”  How this happened, I really didn’t know (except that it involved math).  This ignorance (while not ideal) was not a problem for me in the past because everything worked the way it should.  But this week I ran into an instance where I had a really crappy looking image and deconvolution made it look even worse.  Which was counter to my thinking of what deconvolution was all about.

I now know that deconvolution is a mathematical way of assigning light back to its original source.  Think of a street light.  Normally, when you are looking at a street light, you see a halo of light surrounding it (particularly if it is foggy out).  The same is true for the fluorescent molecules that I am looking at in my sample.  However, I need to see exactly where the object is.  So now, think of two street lights really close together.  If they are close enough, their halos of light may become combined to look like one halo.  Now, if it was so foggy that you couldn’t actually see the street lights themselves, just the halo, you might think there is only one street light there when in fact there are two.  In my images, I really need to be able to tell if I am looking at one object or two objects really close together.  Deconvolution uses mathematical algorithms to get rid of the halo and only let you see the light that’s actually on the object.  It’s like getting rid of the halo on the street light such that you only see the light that is within the street light itself.

Did that make any sense at all?

Anyhoo, deconvolution is important in astronomy, too, for images coming from telescopes.  Because of this, my husband and I had several very long conversations discussing my image processing woes and how I might be able to resolve them (resolve–hahaha!).  Some couples may watch romantic movies on Valentine’s Day, we discuss image processing.

I was really hoping to be able to cross off some of the images on my list (to indicate completion) on the Paper Progress page, but alas it was not to be for this week.  Therefore, the highlight of my week was when a small rose plant arrived in lab for me for Valentine’s Day from my husband.

The Seminar Sock:

This week, the sock and I went to two thesis defenses (for people who are two years behind me in grad school years;  let’s not talk about how I feel about that).  The first one was from a student whose lab studies the mechanism of recombination during meiosis in yeast.  He had some really fantastic electron micrographs of DNA coated with his favorite protein.  He also had worked on an anti-cancer agent and had some interesting data about that.

The second thesis defense was from a student whose lab studies splicesome assembly and disassembly in yeast.  She had a huge amount of data.  Every time she came to a summary, I thought for sure that must be the last thing because it was so much work and then she went on to test another part of her model.  She’s going to have four papers when she leaves.  Amazing.  Her data was not as cool to look at as the first defense because it mostly consisted of a ton of RNA gels (which, all look about the same; I’m all about pretty pictures in science, this is probably why I’m in the lab that I’m in).  But, her method of systematically building her model was very elegant, as was the model itself.

I also have been working on the sock while taking images at the microscope.  I set up the scope to take a series of images.  I can’t leave the room while it’s doing so because then a ton of light would shine on the scope and I need it to be dark.  The computers are not hooked up to the internet, and it’s too dark to read well.  So, I knit.

Plans for next week:

I’m going to finish optimizing this technique if it kills me!  Then, I’m hopefully going to be able to take some images for my paper and cross off some of the figures on my list.

I’m also hoping to post on this blog a series of entries that I’ve been working on that explain immunofluorescence in layperson terms.  I’ve got to do a bit of tweaking on the posts over the weekend, but I should be able to put the first one up on Monday.  I’m also hoping to follow that up (during the next week) with some pictures of an immunofluorescence experiment in progress, culminating in pictures of the slides that I take on the scope.

Stay tuned!

With appologies to Jane Austen

It is a truth universally acknowledged that a new PI in possession of a large grant must be in want of a post-doc.

As previously described

As a bench scientist, there is nothing I hate more about designing a protocol than going to the Materials and Methods section of a publication which used that technique and seeing this:

Because, then, having already taken the time to look up the first paper, you now have to look up the second paper in which sometimes–in an effort to make my brain explode or something–the procedure is cross-referenced yet again.   This can only be made more annoying by this fact:

The paper is from my lab.

Yes, folks, I looked in our book of Lab Methods with no success and I looked in a published book of protocols where this particular protocol was written by my advisor and the critical bit of information I needed was missing.  I then had to find a paper in which we used this procedure only to find the above explanation.  Finally, I tracked down the original paper which was fortunately not too difficult since it was from our lab (but I couldn’t find my copy so I had to get it from pubmed).

Needless to say, I was a little frustrated by the whole experience.  Fortunately, I now have the information I needed and I’m going to start the procedure right now.

Lab Update for Feb. 8

This week I tried planning each day in advance; writing a list of things to do and not going home until I got them all done. As usual when I try this type of thing, I grossly overbooked myself and am now exhausted.  Somehow, I thought it would be a good idea to spend 12 hours a day in lab for 4 out of 5 days.  I was wrong.  Well, it was good in terms of data, but bad in terms of my sanity!

I have added a new page to the blog called Paper Progress which has a very basic outline of the structure of my paper so far.  The first three figures are well-planned in terms of structure, but the layout of last three has not yet been planned, so I didn’t elaborate on them.  As I get the data for each figure, I’ll cross it off my list (I’ve been looking for a widget to do this so I can put it in my sidebar, but so far I have not found anything to my liking).  The titles of the figures in the list are not the titles of the figures in real life.  I have not yet determined how much information about my data I can put on the blog without potentially jeopardizing publication of the final paper.  Some journals would consider my blogging about the data publishing it and therefore refuse to publish my paper on the grounds that it has already been published.  I would prefer to publish in an open access journal that would not give me trouble about such things, but where I publish is not a decision that’s entirely my hands.  So, for now, the descriptions are rather vague.

Mostly, I have been trying to get good images of immunofluorescence data  for Figure 1A.  Because the fluorescence fades after time, if I do not capture good images fairly soon after the experiment is complete, I will have to repeat the experiment.   This week, I did several experiments and then today tried to take pictures of these experiments.  The images are not of the quality that I would like and this cannot be changed by the imaging process, so I need to tweak the experiment.  I’m going to leave that until Monday.

This experiment is a huge pain in the @ss because it requires 12 hours of lab time in one day.  There are very long incubation times and this allows me to do other things during those times.  This included evaluating some of the yeast strains that I created for Figure 2A, prepping DNA to create more yeast strains for Figure 2A, creating the yeast strain for Figure 2B, and optimizing the immunofluorescence procedure for Figure 1B.

I realize this probably sounds like gibberish to most people who are not cell biologists.  Therefore, I’m working on a series of posts about basic concepts and techniques.  The first will be about immunofluorescence, hopefully followed by molecular cloning, and transforming bacteria and yeast.  Stay tuned!

Of Seminars and Socks

Part of being a scientist is attending seminars about current scientific research given by scientists (usually) from outside your university. These are usually about an hour in length. One of the major draws is that they provide coffee and cookies and graduate students can smell free food a mile away.

I am really not emotionally constructed to sit still in a dark room and pay attention to a talk for one hour. After about half an hour, I get very fidgety unless the speaker is particularly engaging. Once I start to fidget, I stop paying attention. I’ve tried taking notes, or drinking more caffeine, or doodling, but none of these things has helped me.

So, I knit socks.

This probably seems counterintuitive to a lot of people. Generally speaking, these are people who do not knit (or crochet, or embroider). People who do knit often tell me how much knitting helps them to pay attention to what’s going on around them. It’s the same for me. It’s as though it keeps the fidgety part of my brain occupied leaving the rest of my brain free to absorb what the seminar speaker is saying (as an aside, I think this would make and interesting study using a PET scan or functional MRI).

Also, if the seminar speaker is deadly dull, or it turns out the talk is completely beyond my comprehension, no harm done. I’ve got my knitting.

It has gotten to the point that I just bring my knitting to every sort of talk/meeting/lecture I go to. Including lab meeting. I tried to be very discreet about it at first, but now I just haul it out and start stitching. My advisor once said, “You know, they sell socks in stores now,” but that’s it. I still contribute to lab meeting. I still even take notes during seminars.

Sometimes, I wonder what the speaker thinks when they see me there with the knitting.  I try to do all of the appropriate things to show that I’m paying attention.  I nod, I frown if I’m puzzled, I laugh when appropriate.  Still, they probably wonder if I’m really taking in anything they are saying.  For awhile, this used to concern me.  I didn’t want to appear rude.  However, I’ve come to the conclusion that it is better to appear rude by knitting and actually get something out of the talk than to not appear rude by knitting and be rude by falling asleep.

So, why socks? Well, socks are small and therefore very portable. And, I only knit on simple ones during seminars. In fact, I have a sock I carry around with me for the sole purpose of mindless knitting while in meetings, at seminars, on the bus, or waiting in line (or at the movie, or at the opera, or at the symphony, or on the plane, or at a thesis defense, or before church starts). And by simple I mean that I can knit on it in the dark with few adverse effects (sometimes I find a mistake that I made and I have to rip back which I do sometime when I can give it my full attention).

My current seminar sock looks like this:

It’s made of hand-dyed wool. I’m knitting it for my husband. I’m about halfway done with this one sock–I still have to knit the foot, including the heel and toe (which I only do when I can pay attention to it) and the whole second sock. I’ll let you know how it progresses.

Misquote of the day

For aught that I could ever read,
Could ever hear by tale or history,
The course of scientific research never did run smooth

–With apologies to Shakespeare