Practical Introduction to Biochemistry

My notes to a physicist friend hoping to join me in a yeast lab

Nov 23, 2020 · 16 min read
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Structure of adenylyl cyclase determined by cryoelectron microscopy. The membrane is shown schematically. (Protein Data Bank)

The lab where I work is interested in the mechanics of basic biological processes, which we dissect using yeast as a model organism.

A good friend, a physicist and technology marketing executive by training and profession, will hopefully be joining me in the lab. These are my informal notes to him to get him up to speed in a practical way for our lab, starting with classical methods. My hope is that he, and others interested in making a career transition into a bio lab, will find this introduction useful as well.

The first installment was on molecular biology which you can find here.

This installment is about biochemistry.

1. Biochemistry

Biochemistry began in the 1800s as a way to study biology and to understand the mechanics of life without the complexity of cells. I also think of biochemistry as illustrating both the powers and limitations of Science’s reductive methods, where we:

  • Break up animals into organs.
  • Break up organs into cells.
  • Break up cells into subcellular components like proteins, DNA, RNA, and lipids.
  • Study the proteins (for example) and its parts in molecular detail — this is the heart of biochemistry.
  • Then reverse the process to verify and validate the new biochemistry data in the context of the cell and the organism.

There are several good textbooks on biochemistry like Lehninger’s Principles of Biochemistry. This link is to an old edition of a biochemistry textbook which happens to be free and in searchable form here.

Wikipedia is a reasonably current and convenient resource for the basics and I will use it liberally here.

If you had to pick one class of biological molecule to study, proteins would be a great choice because of their immense variety and the fact that they do almost all the work in the cell. That variety also makes them challenging (in a good way) to study.

A protein, at its most basic level, is a chain of amino acids. It is much more than that, of course, but we’ll start with that.

2. Analyzing proteins — running a gel

The most basic and practical method for analyzing proteins is called gel electrophoresis. This method sorts the various proteins in your sample by size, big proteins (long chains of amino acids) at the top of the gel, small (short chains of amino acids) at the bottom. Here’s an example of a protein gel:

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Image of SDS-PAGE gel separating proteins of the erythrocyte membrane (Wikimedia Commons)

How do the proteins sort themselves by size in this gel?

They have help in the form of an electric charge. Your protein sample is placed into a well at the top of the gel, and an electric field is applied with a negative charge at the top, and a positive charge at the bottom. The proteins are prepared so they have a uniform negative charge per unit of size. These prepared protein molecules are repelled by the negative charge, and migrate through the gel towards the positive charge at the bottom.

The gel itself is like a porous 3-dimensional sieve. The smaller proteins find it easier to migrate through this sieve, and the larger proteins do so much more slowly, at a rate proportional to their size and charge. This method is generally known as gel electrophoresis.

We already ran into the idea of gel electrophoresis in sorting the size of DNA fragments in the Molecular Biology intro here. The word electrophoresis just means “charge carrier” — and the concept is the same as for DNA. The details of the materials and methods vary to accommodate chemical differences between DNA and proteins. DNA naturally has a negative charge associated with the phosphate groups that connect each nucleoside (sugar-base group). Because there is one phosphate group for each base added to the DNA, the negative charge naturally increases with the length of the DNA.

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SDS-PAGE gel electrophoresis (Wikimedia Commons)

For proteins, the specific type of gel electrophoresis most often used is called SDS-PAGE which stands for: sodium dodecyl sulfate polyacrylamide gel electrophoresis. Let’s break that down.

We already know gel electrophoresis — that is the method we are using to separate proteins (or DNA or other biomolecules) by size.

Polyacrylamide is just the material that the gel is made of. For DNA we often use agarose, which is a polysaccharide material derived from sea weed. For proteins we often use polyacrylamide which is a synthetic gel. It is made by mixing acrylamide and bis-acrylamide. These compounds form a crosslinked polymer network when we add the polymerizing agents, ammonium persulfate (APS) and TEMED (I won’t bother writing that out — you can look it up if you want).

Sodium dodecyl sulfate, also known as lauryl sulfate, is a negatively charged detergent. We add SDS for a couple reasons.

We saw before how DNA naturally has a negative charge proportional to its length. Protein does not have this property. It’s charge varies with the different amino acids which compose the protein, some amino acids are positive, some are negative, and most are uncharged. SDS binds at a fixed ratio (one SDS per two amino acids) and since it carries a negative charge, SDS provides the consistent negative charge proportional to the length of the protein.

Another reason we add SDS is because proteins are mostly compact 3-dimensional globs, and we need to unravel them for electrophoresis to work. SDS denatures the protein from a condensed and compact structure to an open linear chain. There is a form of protein electrophoresis in which the protein is kept in its native 3D conformation — but most of what we will do is denaturing gels, so we add SDS.

Another very important reason to add SDS is that many proteins are insoluble and naturally bind to lipids (like in the cell membrane), or to each other and form a useless precipitate. SDS has a hydrophobic tail which naturally binds to similarly hydrophobic parts of the protein, enabling them to go into solution and to be carried along through the gel.

Once we run the gel and separate the proteins by size, we need to visualize them. We can do that by adding a protein-binding dye to the gel. Common dyes are Coomassie or silver staining.

Here is a video protocol summarizing how to perform an SDS-PAGE experiment.

These gels are also the basis of much more quantitative and powerful methods such as EMSA(electrophoretic mobility shift assay) where we can establish the binding affinity of proteins for their targets.

We’ve talked about gel electrophoresis as an easy way to analyze proteins, sorting them by size. But there is much more to proteins than just size. Many proteins can have the same or similar size, and we need more than a gel to analyze them. Let’s get a little more background on proteins before we talk about other methods.

3. Proteins — some background

Proteins are one of the main building blocks of cells, along with DNA, RNA, lipids, sugars, etc.

Proteins are also the most complex biomolecule, significantly more complex than, say, nucleic acids. Both DNA and RNA are each composed of four different nucleotides, all hanging off a common sugar-phosphate backbone. Proteins, by contrast, are composed of at least twenty different amino acids.

The physical and chemical nature of the amino acids also differ widely from each other, some are positively charged, others are negatively charged, some are water soluble, others are lipid-like (oily) and insoluble, etc. Proteins perform almost all functional and many structural roles in almost all locations within and even outside cells. The variability between proteins is immense. These are some of the factors that complicate the isolation and study of proteins.

To put proteins into context, let’s use Francis Crick’s famous Central Dogma of Molecular Biology, not because it is perfectly correct in all details, which it isn’t, but because it is a powerful framework from which to hang names and pieces of the cell now that we are conceptually breaking it apart.

Francis Crick, of course, was part of the dynamic duo who discovered the structure of DNA. Not necessarily nice duuudes in their treatment of colleagues and especially women. Read about Rosalind Franklin. Science is fundamentally a social and political endeavor whether we like it or not. How we treat our fellow scientists runs to the heart of science. We each need to improve not only scientific knowledge — but also relations with our colleagues.

Once you make a fundamental discovery like Watson and Crick’s, you need to put it into context — what does it mean, this long double-helix DNA molecule with complementary base-pairing… The Central Dogma was Crick’s attempt to put DNA into context.

The Central Dogma paraphrased in its most simplistic cheat-sheet form says: DNA makes RNA makes protein. Crick was more nuanced than this, and although we’re in a hurry, let’s slow it down just a bit and use his Central Dogma to organize the cell’s processes as we know it today.

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Francis Crick’s notes on Central Dogma (Wellcome Library)

Crick’s core insight and focus was on information flow. Biologists at the time had little experimental data to support or refute Crick’s hypothesis, so it is amazing how right he was on many of his broad claims at the time.

The following is what we know today (use your molecular biology textbook to dig into any of these, in as much detail as you like):

  • DNA is composed of four nucleotides abbreviated as A, G, C and T.
  • Triplets (called a “codon”) of these nucleotides encode an amino acid. There are twenty commonly used amino acids in biology.
  • A gene is a long chain of nucleotides which encodes a linear sequence of amino acids. The sequence of amino acids begins to define a protein.
  • A protein is more than a linear sequence of amino acids — it is also the 3-dimensional conformation of that chain. Much of the information for how a protein folds is also encoded within the amino acid sequence.
  • The first transfer of information goes from DNA to RNA (so the phrase “DNA makes RNA” is not quite accurate). This process is called “transcription”.
  • RNA is a nucleic acid like DNA, but less chemically stable, and is composed of nucleotides A, G, C and U (uracil replaces thymine).
  • A complex protein machine called an RNA polymerase transcribes the information from the relatively stable and long-lasting DNA gene, into the more transient, short-lived RNA. The specific type of RNA which encodes proteins is called a messenger RNA or mRNA. This is because the mRNA shuttles out of the nucleus, into the cytoplasm, and to the ribosomes which are the protein-manufacturing machines.
  • The same base-pairing mechanism which allows DNA to specifically pair up with its complementary strand, allows each RNA nucleotide to base pair and faithfully copy the template strand of DNA.
  • The ribosome “translates” the information contained within the mRNA into the long chain of amino acids.
  • A transfer or tRNA has both the triplet code reader at one end, and the amino acid that the triplet codes for, attached at the other. The tRNA, if it matches the triplet on the mRNA, brings the correct amino acid into the reaction center of the ribosome, and the amino acid is added to the growing peptide chain.
  • The final product, the protein, does the vast majority of the functional work (as enzymes) in the cell.

I hope this usefully expands on the cheat-sheet version of the Central Dogma I quoted above.

4. Protein extraction

The rationale for biochemistry is that since cells are far too complex for us to study as a whole with our current technology, we need to extract and isolate sub-cellular components and study those in relative isolation to gain insights into how the cell (and the organism) operate.

So, our first order of business is to extract and isolate proteins from cells.

Since we use yeast, let’s start there.

Yeast, like us, have cells surrounded by a lipid membrane. Our cells are very easy to break open and to begin separating components.

But yeast, unlike us, have a tough cell wall which reinforces the flimsy cell membrane. So, yeast require an additional step that human and other mammalian cells do not: disruption of the cell walls.

We’ll review just a couple of the many different methods to disrupt yeast cells.

One method we use is a simple “bead beating” method to break the yeast’s cell walls. This is where yeast cells are suspended in an appropriate liquid medium to preserve the proteins along with very small glass or ceramic beads. We then shake the mixture in a closed tube so the collision between beads breaks the cells apart.

Another is a chemical method, such as using concentrated sodium hydroxide (or “lye” in household products) and high heat to soften and burst the cells.

One method that I’ve come to appreciate is cryogenic ball milling. This involves instantly freezing the cells in liquid nitrogen, and then using stainless steel balls to mill (grind) the frozen cells at liquid nitrogen temperatures. The fine powder can then be stored in a -80C freezer until it is used for various experiments.

I like the cryogenic method for several reasons.

  • Often, we want to collect proteins in their native conformation (shape) or with their normal associations (meaning with their normal biochemical partners in the cell). The native conformation and associations of proteins are disrupted with many methods. The cryogenic method preserves these.
  • Often, we want the kinds and amounts of proteins as they would be under normal or specified experimental conditions. Many methods deviate from those conditions. The cryogenic method minimizes that disruption. For example, the author of the chemical method notes that the yeast cells remain alive for several minutes in the concentrated sodium hydroxide. Those cells are likely generating proteins associated with extreme stress — and will not be representative of proteins in normal or other experimental conditions.
  • The cryogenic method is quite easy to scale up, and is convenient since the frozen lysate (the product of cell disruption) can be stored at -80C for long periods of time.

Let’s focus on the cryogenic method, as the considerations for this method are similar, though the details may differ.

What we have stored at -80C is a fine whitish powder with all the yeast’s cellular components all mixed together.

In order to do anything with this crude cell extract, we need to re-suspend this powder into an aqueous solution. If we want the protein to be functional, the solution should contain salt near the in-vivo concentration, and sodium chloride does just fine for this purpose.

We need the pH to also be near the normal pH for protein to be functional, so the solution should have a buffer set to about 7.5 or so.

Many proteins require certain heavy metal ions to function, so magnesium chloride is added to provide the necessary ions.

There are very aggressive enzymes in all cells whose job is to destroy old, mis-formed, or partial proteins. These are called proteases. Since we are studying proteins, we need to add chemicals which inhibit proteases.

Detergents are important, too. While there is some subtlety to the reagents above, the detergents added to your protein preparation can have dramatic effects. Some very harsh detergents like SDS mentioned earlier can totally disrupt protein conformation and associations. That may be what you are looking for — if so great. But if you want functional proteins, or proteins isolated with their normal cellular partners, SDS and similar detergents are not for you.

If your later experiments require the protein to be isolated in a functional state, or to be isolated with their normal cellular partners, a mild detergent is necessary. Igepal CA-630 (also known as Nonidet P-40 or NP-40) or Triton X-100 are examples of mild detergents.

Other questions to ask as you design your protein extraction method is, where are your proteins of interest located? Are they in the nucleus, or in the cytoplasm, or in the cell membrane, or in the mitochondria, or elsewhere? These locations can affect the method and materials you use to isolate your proteins.

Once the cryogenic lysate (the lysed cell powder) is resuspended in the appropriate ice-cold buffer, it is spun down in a centrifuge at high speed so that the cellular debris collects at the bottom of the tube, and the soluble proteins remain in the aqueous phase and can be transferred to a new tube.

A small sample of this soluble protein can be run on a gel to check the quality of the lysis.

As mentioned previously, examining only the size of the proteins is not enough to analyze the protein. Let’s look at another important method to begin analyzing proteins.

5. Western blot

The western blot is one of the most common methods for identifying specific proteins.

We saw that an SDS-PAGE gel helps us sort proteins in a sample by size within a gel. The problem is we don’t know if any of the bands represents one of our proteins. Let’s say I am interested in a protein called Cdc25. I know the approximate size of the protein. However, many other proteins have the same size. How do I know whether Cdc25 is one of the proteins in a gel?

We can use the powerful specificity of antibodies for this task. How do we do this?

After we run our protein sample in an SDS-PAGE gel, the first thing we do is to transfer those proteins onto a robust plastic membrane. The gel is rather fragile, and does not hold its dimension well. Membranes made of nitrocellulose or polyvinylidene difluoride (PVDF) or even nylon are often used.

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Western blotting protein transfer (Wikimedia Commons)

Proteins adhere to the surface of the membrane by a combination of hydrophobic and charge interactions. The membrane materials are chosen for their ability to bind all proteins.

An unfortunate side effect of the membrane’s non-specific protein-binding ability is that antibodies — which are proteins — will also non-specifically bind to the membrane. To prevent false positive antibody signals, we need to block further protein binding to the membrane after we have transferred the gel. We can bathe the membrane in a solution of some cheap protein — bovine serum albumin (BSA) or non-fat dry milk are both common inexpensive proteins used to block the membrane.

After blocking, we can now incubate the membrane in a solution containing the antibody specific for our protein of interest. This first antibody is called the primary antibody.

After we probe the membrane with our antibody, we need to detect whether the antibody bound to anything. For this we use a secondary antibody which is attached to some means of visualizing. This can be radioactivity, a chemical color-based reaction, fluorescence, or other means of detecting the primary anyibody.

Detection can be film for radioactivity, various photodetection (digital or film-based cameras) for either fluorescence, luminescence, or color-based secondary antibodies.

6. Immunoprecipitation

We just saw how powerful antibodies are in specifically binding to, and therefore detecting, a target protein. We can use this specific binding property of antibodies for other powerful experimental methods. One of those is immunoprecipitation.

Out of a complex mix of proteins, perhaps all the proteins in a cell, we can use antibodies to specifically pull out the one protein we are interested in. This is called immunoprecipitation or IP for short.

Even better, if we are interested to discover what other molecules our protein of interest binds to, we can use the specificity of antibodies to help us with that as well. This is called co-IP or pull-down when the other molecules our protein binds to are proteins. When we are interested in what parts of the DNA, or chromatin, our protein binds to, the assay is called chromatin immunoprecipitation or ChIP. When we are interested in what RNA our protein binds to, we do something similar to Chip, but RNA immunoprecipitation or RIP.

To pull our protein, and the things our protein binds to, out of a solution of cell extract, the antibody is often bound to beads which make it easy to manage. We can incubate the antibody/beads with the cell extract, then centrifuge the tube such that the beads settle to the bottom and we can easily remove the cell extract and wash the beads of remaining contaminants.

We can then analyze the material pulled down with the beads using gel electrophoresis, western blot, or other methods.

7. Mass spectrometry and other methods

One of the major disadvantages of immuno-methods is that you need to know the protein you are interested in, and based on that purchase or develop antibodies to that protein. The you can use that antibody in western blots to see when or where your protein is expressed in cells.

But what do you do when you don’t know the protein up front? For example, you do a pull-down experiment using an antibody for Cdc25. You want to know what proteins bind to Cdc25 at certain times in the cell cycle. You have several co-IP samples and want to know what proteins are in there. How do you find out?

One method is to use something called mass spectrometry.

Mass spec uses the physics of mass and charge to analyze molecules and identify them based on their predicted behavior in an electric field. This method will identify unknown proteins in a sample.

We won’t say more since these and other physics-based methods to examine proteins at a molecular and atomic level become more specialized and expensive. Structural biochemistry uses methods like x-ray crystallography, or nuclear magnetic resonance, cryogenic electron microscopy, and others to determine the structure of proteins and other biochemicals at atomic resolution to understand their function in the cell.

8. Wrap-up

That’s it for the biochemistry part of this introduction to the bio lab. There is much much more to discuss. For now, we reviewed:

· Gel electrophoresis

· Central dogma as context for biochemistry

· Protein extraction

· Western blots

· Immunoprecipitation

· Mass spec and other methods

As I said for the molecular biology section, this intro doesn’t replace a textbook, or the effort needed to learn the vocabulary and the concepts contained in a textbook. You need that background to be productive in a lab.

This also doesn’t replace training in general lab practices: handling acids and bases, reading MSDS sheets, disposal and recycling, using equipment, etc.

But hopefully this gives a useful background for the hands-on work we’ll be doing in the lab.

I welcome any and all feedback and corrections! A summary like this is bound to contain errors, omissions, or confusing passages. I will happily edit and correct any time, even long after this is published. Thank you all in advance!

And please check out the related articles in this series:

Also, check out this newer one here:

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Husband, dad, scientist, loves to share sciency stuff and goofiness. Please follow me:

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Medium’s center for scientifically-informed content.


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Husband, dad, scientist, loves to share sciency stuff and goofiness. Please follow me:

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