Antigen and Antibody: Celebrity Couples in Science

Immunohistochemistry (IHC)  is a technique that is commonly performed in labs, and is used to identify cells or components of cells for viewing under a microscope. Here are a few sweet examples of IHC:

This picture shows villi from the small intestine, with nuclei stained red. Epithelial membranes are blue, and green labels the rapidly proliferating epithelial layer of the villi and the crypts. I found this picture on a great website called the Cell Image Library. Check it out sometime if you want to be amazed 🙂

This was taken by Jason Snyder, who has a great blog about neurogenesis which you can find here. Granule cell nuclei are labeled red, while astrocytes and radial glia are in white. Beautiful.

Last, here is a picture taken by myself of the dentate gyrus. Green labels nuclei, while red labels dividing cells (actually, dividing cells look yellow because they are both red and green). As you can see, cell division is kinda rare in the brain.

The post today is not focused on how IHC works in general, but rather the biochemistry behind it. So there will be no discussion about the difference between monoclonal and polyclonal antibodies, or what a negative control is, or the difference between primary and secondary antibodies, etc. This post will (attempt) to describe what ACTUALLY happens when an antibody binds a ligand, how EXACTLY a fluorophore works, and what fixation REALLY links.

So first, antibodies and their antigens, the celebrity couple of science. Sometimes they are together, sometimes they break up, but we always will think of them as a pair. Scientists may not know who Katy Perry is currently with, but they DEFINITELY know which antibodies go well with certain antigens, and get very upset when things don’t work out.

An antigen is a characteristic that is unique to the target you are looking for. For example, if we want to label neurons, we would find a component of the neuron that the surrounding cells don’t have.

The antibody is the labeling protein. In other words, it tags the cell you are looking for. Antibodies are very specific and will only tag the cells containing the unique antigen.

The use of antibodies in immunohistochemistry has a very interesting history. Did you know that the reason these antibodies work so well is the same reason why you won’t get chickenpox twice in your lifetime? When you get chickenpox, your body’s immune system creates antibodies that are specifically targeted to the chicken pox virus; that is, the virus antigen. After the virus leaves, your body still has those antibodies which are ready to fight the next time chickenpox tries to attack.

Antibodies for immunohistochemistry are made very similarly. The protein that you want to detect is first injected into a rabbit. Since this is not natural (just like chickenpox), the rabbit has an immune response and creates antibodies specifically targeted to the protein. Those antibodies are then collected from the rabbit and purified. Scientists then apply those antibodies to the tissue they are studying. I find it amazing that scientists have turned an already amazing phenomenon which saves people’s lives into a research tool which can then save more lives.

The main types of interactions that hold antigens and antibodies together are van der Waal forces, electrostatic interactions, and hydrogen bonds. No covalent bonds are formed when antibodies bind antigens. Van der Waal forces grow stronger when the distance between the antibody and antigen is reduced. When the structure of the antibody fits well with the antigen, the distance between them is shortened, allowing van der Waal forces to play a part. The same is true for electrostatic bonds. In electrostatic bonds, opposite charges attract. The amino acids arginine, histidine, lysine, aspartic acid and glutamic acid are electrically charged, so any protein with these amino acids can form electrostatic interactions with another charged protein.

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Interesting stuff!

One last bit of trivia: Temperature is an important factor in the association of antibody and antigen. This can be seen in an equation for the acid dissociation constant, KA =eΔaG/RT !

So that is just a little about the biochemistry behind IHC. Below are the sources that I used to create this post. If I have made any mistakes, I apologize, as I am learning this as I go along 🙂

  • Boenisch. (2001) Handbook Immunochemical Staining Methods. 3rd Ed. DAKO
  • Atassi, van Oss and Absolom. (1984) Molecular Immunology: A Textbook.

How to Get Rid of Skid Marks.

In this post I hope to review the basics of enzymes and their application to laundry detergents, products that we (hopefully) use often. One of the first things I did in preparation for this blog was look at the ingredients in my own laundry detergent, all® small & mighty with stainlifters.

I looked at the list of ingredients on the back and….. I was very disappointed. “Cleaning agents (anionic and non ionic surfactants), buffering agent, stabilizer and brightening agent.”


Anyway, there are some laundry detergents that use enzymes, including Tide, Arm and Hammer, and Ultra Plus, which you can read about here. So what kind of enzymes are used in laundry detergents? They typically include proteases, lipases, and amylases . Some detergents also include cellulases and peroxidases, which remove soil and brighten colors, respectively. The list below was modifed from this paper if you want to read more.

Protease: Breaks down protein. Common protein stains include blood, sweat, egg, and grass.

Lipase: Breaks down lipid, AKA fat. Common stains are from butter, oil, and salad dressing.

Amylase: Breaks down starch-based residues found in food such as spaghetti, custard, chocolate, gravy, and potatoes.

Cellulase: Removes soil indirectly by breaking down cellulose. Used on cotton fabric, as it does not break down the cotton fibers.

Peroxidase: Bleaches dye that is released from fabric to prevent bleeding onto other fabric.

Before we discuss how proteases, lipases, amylases, etc. do their thing, let’s talk about enzymes in general. For all the reactions described above, energy is required. The more energy required, the slower the rate in which it proceeds. What enzymes do is reduce the energy required for the reaction to occur. For example, a protease reduces the energy input needed to break down a protein. Once the enzyme is added, the energy needed is lower, allowing the rate of the reaction to proceed. It’s kind of like having a sherpa with you when you climb Mount Everest.

Another important fact about enzymes is that once a reaction is done, the enzyme is still available to catalyze another one. In other words, a protease molecule can break down more than one protein. This is one of the main reasons why only a small amount of enzyme is needed to make a big effect in laundry detergent, something that producers definitely like.

Other reasons why laundry detergent companies like enzymes are

  • they are cheap
  • they have specific actions
  • they send less organic pollutants into the water
  • they are non-corrosive

Enzymes are relatively cheap because they can be made easily in large quantities, as long as you know a little about genetics and cell culture. Enzymes are “made” by growing cells that are known to produce a lot of the enzyme  of interest. The types of cells often used are yeast, bacteria, or fungi. Once the cells have grown, they can be broke open and the enzyme you want can be isolated. Scientists have improved upon this method by altering the cells’ genome, causing the cells to grow abnormally high amounts of the enzyme desired.

A suitable enzyme for use in laundry detergents must also have the following characteristics:

  • compatibility with detergents (the actual detergent in “laundry detergent” can denature proteins, as we talked about last week in The Incredible Edible Egg. Since enzymes are proteins, they must be resistant to the detergents used to clean fabric)
  • must work at a variety of temperatures
  • must be stable at a pH range from 8 to 10.5

The ability of enzymes to work at a variety of temperatures, especially low temperatures, makes laundry detergent more environmentally-friendly. It also can replace old ingredients used in laundry detergents that are harmful to the environment.

Let’s select protease and lipase to see how they work.

Since we talked about eggs last week, lets pretend you got egg white on your shirt. The main protein in egg whites is albumin. So how does a protease break down albumin? Most commercially used enzymes are alkaline serine proteases (see this paper for a review about alkaline proteases). Serine proteases use 3 amino acids that work as the Three Musketeers also known as the catalytic triad to break bonds in albumin.

  1. The protease and albumin bind, forming a Michaelis complex. The portion of albumin binding to the protease is shown in red.
  2. Serine (Ser 195)  from the enzyme attacks a bond in the protein albumin which connects its subcomponents, or amino acids, together. This attack causes the formation of a tetrahedral intermediate.
  3. The tetrahydral intermediate changes into an acyl-enzyme intermediate due to general acid catalysis from histidine (His 57). The albumin bond has been broken, but albumin is still bound to the protease.
  4. One part of the broken albumin protein is released.
  5. The acyl-enzyme intermediate is deacetylated, forming a second tetrahedral intermediate.
  6. The other half of the broken albumin protein is released. The protease is left in its original form.
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Once you finally got that egg stain off your shirt, you had to be a klutz and smear some butter on it. What would you use to remove butter? Butter has a high amount of lipids called triacylglycerol, so a lipase would be a good enzyme to use.

Berg JM, Tymoczko JL, & Stryer L. (2002) Biochemistry. 5th Ed. W.H. Freeman: New York, NY.

It is crazy to think that all of this is going on every time you run a load of laundry. Pretty sweet! As a reward for getting through all of these boring diagrams, here are some awesome pictures and video that won the 2011 International Science and Engineering Photo Challenge.

Metabolomic Eye, Bryan W. Jones (Photography – 1st Place)
Science 3 February 2012: Vol. 335 no. 6068 pp. 526-527


The Ebola Virus, Ivan Konstantinov et al. (Informational Posters & Graphics – Honorable Mention)
Science 3 February 2012: Vol. 335 no. 6068 pp. 530-531

BTW, getting the Ebola virus is my worst fear. Ever. But it sure looks cool!

Powers of Minus Ten, Laura Lynn Gonzalez (Interactive Games – Honorable Mention)
Science 3 February 2012: Vol. 335 no. 6068 pp. 532-533

Well, I hope you have a great day! I am off to play Powers of Minus Ten. 🙂

The Incredible Edible Egg

Today’s post will be focused on the biochemistry of cooking, AKA molecular gastronomy. I never realized how much research exists behind cooking. Check out Gastronomica: the journal of food and culture for some interesting food articles. Another great source I found was the book “On Food and Cooking: The Science and Lore of the Kitchen” by Harld McGee. Lastly, the Bible of biochemistry, “Fundamentals of Biochemistry: Life at the Molecular Level” by Voet, Voet and Pratt will be referenced.

There is a lot about eggs to study: Why do chicken eggs have hard shells? What makes eggs different colors? How do the egg yolk and white differ in terms of nutritional content? What happens when you cook an egg? We will focus on this last question today, specifically, the cooking of egg whites. Here’s a fun fact: a search on PubMed today for “egg white” resulted in 5,280 articles!

The basic principle behind the cooking of egg whites is the denaturation, or unfolding, of egg white proteins. When the proteins are properly folded, egg whites look clear, like this:

Proteins fold due to the properties of protein subcomponents called amino acids. Amino acids are the building blocks of proteins. Some amino acids do not like water; this is called hydrophobicity. Other amino acids are attracted to water; that is, they are hydrophilic. The technical term defining a protein’s hydrophobic and hydrophilic tendencies is called hydropathy. When in water, the protein will conform itself so that the amino acids with hydrophobic, or “water fearing”  characteristics are tucked inside the protein. Meanwhile, the amino acids with hydrophilic, or “water loving” tendencies will orient towards the water. Other factors that determine protein stability are electrostatic forces and disulfide bonds. We will talk about disulfide bonds in a little bit.

When egg white proteins are heated, they start to unfold.  Next, the unfolded proteins start to clump together, forming a network of protein. This is what coagulates, or solidifies, the egg white, making it look something like this:

A variety of forces can cause proteins to denature, including heat, pH, detergents, and chaotropic agents. We denature our egg white proteins by heat (For a cool video on the denaturation of egg whites using acid, click here. I suppose it would taste pretty normal once you rinsed off the acid, but I wonder about salmonella…). Egg whites contain a variety of proteins, the top three being ovalbumin at 54%, ovatransferrin at 12%, and ovomucoid at 11%. Each of these proteins denatures at a different temperature: ovalbumin at 84.5 degrees Celsius (about 184 degrees Fahrenheit), ovatransferrin at 60 degrees (140 degrees Fahrenheit), and ovomucoid at 70 degrees (158 degrees Fahrenheit).

As I said earlier, once egg white protein denatures, the proteins aggregate together, changing the egg white from a clear, viscous liquid to a white solid. I would assume that the proteins are driven to each other because the hydrophobic amino acids are exposed to water and don’t like it, but I could be wrong. The type of bond that the proteins form with each other are disulfide bonds, as determined in this paper by Mine et al. Disulfide bonds are formed between the thiol groups of cysteine residues, a component of certain amino acids.

If you heat your eggs to 84.5 degrees, the melting point of ovalbumin, the egg whites will solidify and become rubbery. So apparently, we don’t like all of our ovalbumin denatured in our eggs. The recommended temperature to cook egg whites is about 70 degrees, which would denature some, but not all of the egg white protein. Mathematician Douglas Baldwin has done a lot of studying behind the science of cooking an egg perfectly. In fact, he figured out the math behind cooking an egg to have slightly firm egg white while keeping the yolk liquid, which you can read here if you so desire.

J. Kenji Lopez-Alt also has a great post at in which a bunch of experiments were performed with eggs. This picture taken from the post shows what happens to an egg when you lower it into boiling water:

I’d like to leave you with a photo of my absolute favorite breakfast food: Eggs Benedict 🙂

Thank you for stopping by my blog, and have a wonderful day!