Optimizing a Western Blot

In the last post, we talked about the process of performing a Western blot, and a lot of the steps require optimization.  Exactly why is that?  How can you optimize these steps?

But why?!
Let's start from the beginning.  Why do we need to optimize Western blots at all?  Western blots rely on antibodies to detect proteins, and antibodies are created in animals.  This process involves purifying a protein (or a chemical), injecting the antigen (protein or chemical) into an animal, collecting that animal's serum, and purifying antibodies.  This process produces polyclonal antibodies, and the purification process can affect their specificity.  For many scientists, raising one's own antibodies is laborious and unnecessary, as many antibodies are commercially available.  If an antibody is not commercially available, one can purify a protein and send it to a company in order to produce an antibody.

The above process results in polyclonal antibodies.  Monoclonal antibodies can be derived by isolating B cells from stimulated animals, detecting which B cell population is producing the antibody against your antigen, and growing this B cell in culture.  Of course, more steps than this are involved, and the previous sentence is a very rough description.  Regardless, using this method, one can make virtually unlimited supplies of antibody.

Importantly, no two animals will react to an antigen in the same way - that's the beauty (and difficulty) of the immune system.  Thus, no two antibodies are really the same either - unless you have monoclonal antibodies.

To summarize:

• Western blots rely on antibodies for detection
• Creating antibodies is technically difficult and involves injecting an animal with an antigen
• Polyclonal and monoclonal antibodies can be produced and they have different uses
• Different animals produce different antibodies
• Animals within the same species will make slightly different antibodies.

Using antibodies

Antibodies are used for many processes in biomedical science: Western blots, immunofluorescence, ELISA, immunoprecipitation...  If an antibody works (or doesn't work) for any of these processes, it may (or may not) work for another.  Prior to use, antibodies must be tested and optimized.

The steps that require optimization

Let's review the steps in performing a Western blot that require optimization

• Blocking agent
• Detergent concentration
• Antibody concentration
• Incubation times and temperatures
• Secondary antibody concentrations, incubation times and temperatures
• Exposure time

We'll go through these one at a time.

The blocking agent
In Western blotting, two blocking agents are typically used: non-fat milk or bovine serum albumin (BSA).  Typically, milk is used because it is cheaper than BSA.  Typical concentrations of milk range from 5-10% (weight by volunme).  However, for antibodies directed against phosphoproteins, BSA must be used because these antibodies will be neutralized by milk.

In addition to which blocking agent to use, one can also optimize the amount of time for blocking and the temperature.

In my personal experience, 5% milk in 2% TBST (Tris-buffered saline with Tween) for 1 hour at room temperature does the trick.  If you're in a rush, 30 minutes at 37 degrees can also work.

Detergent concentration
In Western blotting detergents are used to remove excess antibody and to prevent background noise.  Generally, a low concentration of detergent is sufficient to clear antibodies binding non-specifically to a membrane.  For me, 2% Tween works well, and it can be diluted in Tris- or phosphate-buffered saline.  For antibodies with greater background noise, the concentration of Tween can be increased, or the number / length of washes can be increased.

Antibody concentration
Probably the best place to start with optimization with with how much antibody you're using.  Naturally, you want to use less antibody (they're expensive, after all!), but it's good to try a range of concentrations.  For me, 1 uL of antibody in 1 mL (1:1000) is a good starting point.  Some antibodies work at 1:20,000; others work at 1:100.  It entirely depends on the antibody, and the manufacturer should provide guidelines for what concentrations to try.

Also, keep in mind that the concentration of your antibody depends on the application:  one concentration for Western blotting may not work for immunofluorescence.

Incubation times and temperatures
Yet another step that requires optimization is how long and how hot to incubate your membranes.  Sometimes, antibodies work quickly and you cannot incubate for long periods of time because this will result in higher background.  However, other antibodies will require longer incubations, even overnight, in order to see any bands.  Generally, longer incubations are done at 4 degrees, while shorter incubations (up to a few hours) can be done at room temperature.  You'll never know exactly what incubation time to use until you try.

Secondary antibody concentrations and incubations
As addressed above for primary antibodies, the same should be done for secondary antibodies - sounds like a lot of work, no?  The good news it that secondary antibodies are generally "well-behaved."  For instance, one particular type of secondary antibody uses a concentration of 1:20,000 in milk or BSA for an hour at room temperature.  No optimization required if you know what the conditions are!  However, if you're working with a new secondary, especially if it's a new species, optimization is your best bet.

Exposure time
One of the last steps to optimize is how long to expose a blot before developing it.  Modern technologies have attempted to supplant this step: new chemiluminescent detectors have reduced our need to measure how long to expose our blots.  However, for those of us that can't afford this fancy new equipment, we may rely on the good old film and developer.  Using this methodology, the amount of time film is exposed to your completed blot will affect how dark your bands are, as well as how much background you.  Of course, you want to optimize this step so that your image is clear and not misleading.  Overexposing a blot can lead to bands that all look the same; in reality, they might not be if you were to expose your blot less.  Additionally, taking several exposures - at both short and long lengths - will give you a range from which to choose.

Conclusions and ideas
The above gives a general guide for how to optimize a Western blot, but by no means is it exhaustive.  You can also optimize your developing reagent, for instance.  The best bet is to try something and tweak as needed.  Western blots are truly an art, and they can be really, really frustrating.  However, a little bit of effort in optimization will save you a lot of headache down the road...

How a Western blot works

One of the most important techniques in molecular biology is the Western blot, which is used to detect proteins in a sample.  Running a Western blot can be an intimidating experience, as there are multiple steps, and if care isn't taken at each of the steps, the end product can be reduced in quality.

Several sources exist that explain in more technical detail how to run a Western blot, including this article from the National Institutes of Health.  Below is a summary of the theory and basics of how to run a Western blot.

The Sample:  To run a Western blot, we need some kind of sample, which in most cases is a cellular lysate, which we want to probe for the presence (and possibly abundance) of a protein.

Cell lysates from tissue culture can be collected by removing growth medium, washing in a neutral solution (such as PBS) and adding a lysis buffer to break open the cells.  Additionally, tissues from mouse organs or even human samples can be ground into lysis buffer, or bead-beaten into small pieces.  The lysed cells will need to be broken up in order to load them into our Western blot and in order to develop a nice final product.

After cell lysis, we've got two things to do.  First, we need to figure out how much protein we've got in the sample.  Determining protein concentration is usually done with a Bradford assay, which is a colorimetric assay.  A future post will deal with the details of this assay.

Next we also have to destroy DNA in the sample, while reducing disulfide bonds in our proteins.  Typically, this is done with a buffer containing beta-mercaptoethanol (the stinky stuff!) and boiling.  Protein samples are usually very viscous due to DNA, and if boiling doesn't get rid of all of the viscosity, a syringe and needle can be used to mechanically shear the DNA.

Running the Gel:  Many labs now use pre-made acrylamide gels, but many make their own gels as well.  A future post will have to delve into the details of what types and concentrations of gels to use.  For now, let's assume that we have the right kind of gel, the right percent acrylamide, and we've received the gel from a company.

In order to estimate the size of your protein product, a molecular weight marker should be run alongside the sample.  These ladders are commercially available and allow for approximation of molecular weights.  Being able to tell the size of a protein is important - sometimes you can be surprised by what size your protein looks compared to what you had expected!  If a protein is running at a "weird" size, don't shrug it off - that could mean something important.  In addition to running your samples and molecular weight marker in the gel, you want to fill every empty lane with buffer.  This will aid in running the gel and prevent proteins in your sample from shifting to the empty side of the gel.

Polyacrylamide gels are typically run at about 100-200V for anywhere from 30 minutes to three hours, depending on the size of the protein and the resolution you would like.  But what's really going on when the gel is running?  The buffer used to lyse cells contains sodium dodecyl sulfate (SDS), which is a negatively charged molecule (and it's an irritant, so always be careful when working with it), and molecules of SDS cover the proteins in the sample.  Since SDS is charged, applying a current to a gel loaded with protein covered in SDS causes this protein to migrate through the gel.  Bigger proteins (or bulkier proteins in general) move through the gel more slowly, so they don't migrate through the gel as quickly.

When a voltage is applied to the gel, the proteins migrate through two phases of the gel: the stacking layer and the resolving layer.  The stacking layer orders the proteins by length, based on their charge (from the SDS) and the resolving layer then expands the distance between these proteins, resulting in a fully resolved (and readable) gel.

After running the gel, it's time to transfer!  Transferring a Western blot involves moving your resolved proteins in the polyacrylamide gel into a membrane, usually PVDF or nitrocellulose.  This process can be done in the old school manner - by capillary action.  To use capillary action, the polyacrylamide gel is placed below the membrane, which is then stacked with paper towels.  The paper towels absorb moisture and draw proteins from the gel into the membrane, where they are "stuck."  This process is usually done overnight; thus, to reduce the amount of time this transfer takes, most researchers use either a wet or semi-dry transfer apparatus to use a current to draw proteins from the acrylamide into the membrane.

Following transfer, it's time to start to probe for our protein of interest.  Here is where Western blotting becomes an art:  everyone seems to do this step differently, and how this step is done depends on the antibody as well.  Regardless, there are three general steps.  First, we must block the membrane with nonspecific proteins, which is usually done with non-fat milk or with bovine serum albumin fraction IV (BSA).  The membrane is incubated in a solution for a specified amount of time at a specified temperature (such as 4 degrees, overnight or 37 degrees for an hour).  This process coats the membrane and prevents your antibody from binding non-specifically.

Next up:  primary antibody.  The primary antibody is the expensive reagent you can purchase from a number of vendors.  Antibodies can be easy to use, or they can be difficult.  They can be raised in mice, rats, chickens, goats, and more.  They can be monoclonal or polyclonal as well.  Thus, there is a lot of variability in antibodies, and your experiences with an antibody may be completely different from any other antibody.  This is part of the art of Western blotting: it is necessary to try things and to optimize your protocol.  Your membrane will be incubated with primary antibody, again for a specified amount of time, at a specified temperature and at a specified concentration.

Following incubation with the primary antibody, the membrane is washed in a solution containing a low amount of detergent (such as Tween), and then it's time for the secondary antibody.  The secondary antibody binds to the primary antibody and also contains some sort of means of detection - fluorescence or horse radish peroxidase (HRP) activity, for instance.  By using a secondary antibody, we greatly increase the specificity of the assay - in order to detect our protein of interest, that protein must be bound by the primary and secondary antibody.  The same conditions for the secondary apply as for the primary - one must figure out the best conditions for the antibody given the needs of the assay.

Immediately after incubation with the secondary antibody is detection - when things get interesting.  For the purposes of keeping this post short (kind of!), we'll describe the old-school method of exposure the blot to film.  After washing the excess secondary antibody off the membrane, one way to detect our protein is to use reagents that emit light via the HRP activity of the secondary antibody.  The detection reagent is added to the membrane for a short period of time and then removed.  Then, the membrane is moved to a dark room where the membrane is exposed to film for a specified amount of time.

After exposure, we need to develop the Western blot, by running the film through a developer.  Here's where we obtain our final product - a film with lines and smudges indicating (hopefully!) that our protein is where we hope it is.  At this point, we will know how much more optimization we need to do.

As you may have noticed, there is a lot of optimization of Western blots.  Below is a short list of steps that can be optimized:

1. Running conditions of the polyacrylamide gel (percent polyacrylamide, voltage)
2. Type of membrane - PVDF versus nitrocellulose
3. Transfer conditions - wet, semi-dry, voltages, times
4. Blocking conditions, times, and temperatures
5. Membrane wash components - amount of detergent, number of washes
6. Primary antibody incubation conditions  - times, concentrations, and temperatures
7. Secondary antibody incubation conditions - times, concentrations, and temperatures
8. Detection method
9. Exposure time (if applicable)

Needless to say, Western blotting is an art.  The optimization steps must be done for every single antibody, which can be difficult, especially if the antibody is particularly difficult to work with.  Regardless, Western blotting is a very powerful and popular technique to detect proteins.

Protein Structure II: Digging Deeper

In the previous post, we discussed the different levels of protein structure.  Here, we will consider more specific details of what makes up protein structure.

Previously, we talked about how secondary structure consists of alpha helices and beta sheets, but there are even more nuanced forms of secondary structure.  For example,  different types of alpha helices exist:  the 3.613 and 310 helices are different types that consist of 3.6 and 3 amino acids per turn of the helix, they also have different radii and lengths.

An interesting little tidbit about alpha helices is that there are certain amino acids that are more likely to be found in helices.  These include alanine, glutamine, leucine, and methionine.  In contrast, there are some amino acids that do not really fit into a helix: proline, glycine, tyrosine, and serine.  These amino acids have structural characteristics (such as proline's "kink") that tend to break helix structure.

Additionally, beta sheets have different properties depending on their orientations.  For example, with a parallel beta sheet, the chains of the protein are oriented in the same direction.  In contrast, anti-parallel beta sheets have chains that run in opposite directions.  See the illustration for a more visual explanation of this trait.

In addition to beta sheets and alpha helices, a few more "secondary structures" exist.  I put this in quotation marks because these types of structures are perhaps not classical structures - in that they're not alpha helices or beta sheets.  The first is the loop, which is a stretch of amino acids that makes a loop - rather self-explanatory!  These loops are important for protein structure because they allow for the creation of relatively compact proteins.  In particular, loops can be found in anti-parallel beta sheets, connecting the two beta strands to make the sheet.  Sometimes these structures are called hairpins because they are tight stretches of amino acids that hold a protein structure together.

The last secondary structural element we'll consider is the crossover loop, which is similar to the hairpin loop, but it connects portions of a protein at a longer distances.  These crossovers can be found especially in anti-parallel beta sheets, connecting the two strands such that they can be anti-parallel.

As mentioned, secondary structure consists of small tracts of protein structure, primarily formed of alpha helices and beta sheets.  These secondary structural elements can be organized into specific combinations called motifs.  Motifs are commonly-found structural organizations in proteins, such as zinc finger or coiled coil motifs.  In the case of a zinc finger motif, there are two beta strands and an alpha helix, making up a fold that looks something like a finger.  Within this motif is a zinc ion, hence its name.  Several proteins contain this motif, which is primarily involved in DNA- and RNA-binding.

Another level of structure is the domain, which is considered a module of a protein.  In general, domains have functions that can be separated from the protein as a whole.  Domains are typically large pieces of proteins (think of them as a swing set on a playground - the playground is for kids to play, and the swing set has its own specific function, to swing!).  An interesting aspect of domains is that they can be found in multiple proteins with similar functions.  For example, some proteins have kinase domains to help with phosphorylation; some have RNA-binding domains; and so on.  By detecting domains within a protein, we can infer its function, and this ability has been incredibly useful in predicting the function of new proteins.

The Basics of Protein Structure Part 1: The Levels of Structure

In a previous post, we had explored the characteristics of the 20 amino acids that make up proteins.  These amino acids make up a diverse collection of molecules that can be strung together, making up proteins that have a multitude of functions within the cell.

Amino acids form a protein through the action of the ribosome, which we will discuss in a future post.  At this time, suffice it to say that the ribosome uses an elegant mechanism to read mRNA and translate it into the protein encoded by the mRNA by adding amino acids in a string.  As this string of amino acids is created, it begins to form a structure that will have functions within the cell.

Four levels of protein structure exist:

Primary (1^o) structure:  The order of the amino acids is the primary structure.  Think of the primary structure as the alphabet of the amino acids: MGRYNVPL, for example.  The primary structure describes what order amino acids are in, and even though it might not seem like very much information, even the primary structure of a protein can provide a great deal of information in terms of its function and its potential 3-dimensional shape.

Secondary (2^o) structure:  When amino acids are polymerized, they form local structures, which make up the secondary structure.  Think of secondary structure as the shape of a group of amino acids.  Two primary forms of secondary structure exist:  alpha helices and beta sheets.  Alpha helices result from the coiling of the amino acid string turning about itself.  In contrast, beta sheets are flatter and lack coiling.  There are several types of alpha helices and beta sheets, which we will save for a future post, and these different types of structures have important implications for overall shape of a protein.

Tertiary (3^o) structure:  Protein structure gets exciting when you talk about tertiary structure, which can be described as the overall three-dimensional shape of a protein.  In general, the tertiary structure is the “final” form of a protein, although modifications on the protein, as well as interactions with other proteins can affect this structure.

Quaternary (4^o) structure:  When proteins interact with each other, they can form complexes, which is the quaternary structure of these proteins.  You can think of this structure as the way proteins contact each other.  The bundling of proteins together can be between proteins of the same type (such as is the case with hemoglobin) or other types of proteins.

The drawing attempts to illustrate the concept of the different levels of protein structure.  Again, think of the primary structure as the order; the secondary structure as the local shape; the tertiary structure as the overall shape; and the quaternary structure as the way this tertiary structure of the protein interacts with other proteins.

How protein structure is established is a fascinating question and a field that is actively studied by prominent labs around the world.  Protein folding is the process of a chain of amino acids curling into its final shape, and how this process occurs is complex and not completely understood.  In general proteins fold depending on their environment (exposed to water or not, for example) and with the help of other proteins, called chaperones.  Protein chaperones help to establish a protein’s structure as well as maintain it during times of stress.  Further, modifications on proteins can change their structures, such as when p53, a protein that is involved in regulating many processes within the cell, is phosphorylated – its structure and, consequently, its function is altered slightly. 

The structure of proteins has fascinated scientists since we first learned about proteins.  Thousands of scientists around the world are still working to discover new structures and to learn how proteins fold.  The science behind protein folding has important implications in diseases such as Alzheimer’s, cancers, and infectious diseases.   

The Basics: Prokaryotes versus Eukaryotes

Biology is an amazing complex topic, and this complexity is what makes it fun!

Before jumping into advanced topics, however, it is important to have a solid foundation of the basics, which we usually learn before algebra even.  As a quick review, let's talk about prokaryotes and eukaryotes, the two types of cells that should be familiar to most everyone.

The world of living cells is broken down into two major types of cells: prokaryotes and eukaryotes (also called procaryotes and eucaryotes, depending how you would like to spell).  Evolutionarily, we consider prokaryotes to be the ancestors of eukaryotes, which we will discuss further in the future.

Let's start with the prokaryotic cell and its defining characteristics:

• Cell wall:  Prokaryotes have a cell wall, which are tough boundaries that enclose the cellular contents.  The cell wall is pretty tough, too - its composition gives it rigidity that keeps the prokaryotic cell's shape.  The cell wall is covered on the outside by the outer membrane and on the inside by the plasma membrane.  
• Nucleoid:  DNA in prokaryotes isn't organized quite the same as in eukaryotes.  Rather than being organized in a distinct, membrane-bound portion of the cell, prokaryotes organize their DNA in what is considered the nucleoid.  You can think of the nucleoid as a membrane-less compartment where the organism's DNA is found.  Although in the diagram I have drawn a single linear DNA molecule, prokaryotes have a diversity of types of DNA, from single circular DNA to multiple linear and circular DNA molecules.
• Cytoplasm:  The area within the cell wall and membranes is considered the cytoplasm - the fluid portion of the cell.  By no means is the cytoplasm empty space - it is filled with molecules and many events are taking place constantly within the cytoplasm.

Prokaryotes may seem "simple," but they certainly are not.  The above points are a simplification, but are the general characteristics of prokaryotes.

Eukaryotes can be considered more "complex," though certainly not better.  As with prokaryotes, the below characteristics are general and apply to most eukaryotic cells.

• Nucleus:  Unlike prokaryotes, eukaryotes have a define space for their DNA - the nucleus.  The nucleus is surrounded by the nuclear membrane, which is surrounded by the endoplasmid reticulum (discussed next).  The nucleus is a hub of activity in the eukaryotic cell and by its enclosure in the nuclear membrane, distinct events can occur here and not in the cytoplasm.
• Endoplasmic reticulum:  Surrounding the nucleus is the endoplasmic reticulum (ER), which is a compartment involved in moving molecules in and out of the nucleus, as well as regulating gene expression.  The ER can be broken down into the smooth and rough ER.  The rough ER is where proteins are made on the membrane.  In contrast, the smooth ER can be considered where pieces of the membrane are exchanged with the Golgi apparatus (next).
• Golgi apparatus:  The Golig consists of a stack of membrane-bound vesicles, which are mainly involved in protein trafficking.  Here, a number of modifications can be made to proteins to target them to specific parts of the cell, such as the membrane or lysosomes.
• Secretory vesicles:  Some molecules in the cell need to be sent outside the cell, and they can exit through secretory vesicles.  These vesicles often originate in the Golgi, after which they fuse with the plasma membrane and dump their contents in the extracellular space.
• Lysosome:  The trash compactor of the cell, the lysosome is involved in recycling the contents of the cell.  Lysosomes have contents that break down molecules into their components so that the cell can reuse them.  Additionally, lysosomes can be used to destroy invading bacteria and to break down molecules that can be harmful to the cell.
• Mitochondria:  "The powerhouse of the cell" - the mitochondria (singular mitochondrion) are involved in energy production, yes, but they also function is several other aspects, such as cell death.  Mitochondria themselves are like miniature cells within the eukaryotic cell, and they have their own DNA (mtDNA).  Mitochondria have many interesting aspects that will be discussed in a future post.
• Plasma membrane:  Similar to prokaryotes, the plasma membrane in eukaryotes acts as a boundary, separating the cell from the outside.  Unlike prokaryotes, however, eukaryotes (generally) do not have a cell wall.  The plasma membrane is a very dynamic part of the cell - signaling, budding, engulfing.  

As mentioned, the above characteristics are an incredible simplification of prokaryotes and eukaryotes, but let this serve as a starting point into learning about each type of cell.  Both types have their own special attributes and many mysteries that remain to be solved.