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Know Your Western Blot Jargon! A Quick Review.

Posted in: Protein Expression and Analysis
Know Your Western Blot Jargon! A Quick Review.

It has been said that “Incomprehensible jargon is the hallmark of a profession” (Kingman Brewster, Jr) and while I cannot speak for other professions, as a biologist I am inclined to believe it.

So whether you need to cover your qualifying exam bases, want to avoid looking like an idiot to your coworkers, or need to explain Western blotting to that new undergrad – you need to know your Western blotting basics and you need to know the jargon that goes with it! Lucky for you, I put together this quick review of SDS-PAGE (Polyacrylamide Gel Electrophoresis) Western blotting including its required jargon (see bolded and hyperlinked words).

The Purpose of a Western Blot

The goal of a Western blot is to acquire qualitative or semi-quantitative information about your protein-of-interest by indirectly visualizing it with an antibody detection system. In short, a Western blot is done by extracting proteins from your cells or tissue and resolving these proteins by gel electrophoresis before transferring them to a membrane where they are probed/blotted with antibodies against your proteins-of-interest.

Sample Preparation

In cells or tissues, your protein-of-interest may be found anywhere and everywhere. So make sure that you understand your choice of extraction buffer, and remember your limitation: That you may only extract/solubilize a fraction of your protein. After extracting as much of your protein as possible you must prep them for electrophoresis. This is typically done by mixing them with sample/loading buffer that contains SDS, glycerol (for ease in loading) and dye (to visualize sample loading). The most important ingredient in this loading buffer is the SDS (sodium dodecyl sulfate), which is an anionic (negatively charged) detergent that partially denatures and coats your protein. To ensure all (well at least most) proteins are fully denatured and have an even coating of SDS, the sample/loading buffers are also sometimes boiled and reducing agents are often added to break cysteine bonds. After this is done, your proteins with their uniform negative charge are ready to be separated, via electricity, by size. But first, you need a gel.

Chemistry of the Gel

There are many websites that cover the nitty-gritty chemistry and polymerization dynamics of acrylamide gels. But in practice, there are only a few things you need to know: There are two parts to an SDS-PAGE gel. The first part, the larger and lower gel portion, is called the resolving gel and it is poured first. Once the resolving gel has polymerized (solidified), the second part is poured on top: the stacking gel. The basic chemistry of these two gels is identical. Instead, they only differ in their acrylamide concentration and pH.

You can buy pre-poured gels or make your own. If you decide to make your own, here is the list of ingredients in a standard Tris-glycine SDS-PAGE gels and an explanation of what they do and why. If you decide to NOT make your own, you should still read this list — after all, you should know the chemistry behind your science!

  • 30% acrylamide mix. This solution is a mix of two different molecules: a linear polymer-acrylamide and a crosslinker-Bis. When your gel’s polymerization reaction is initiated, this mixture forms a lattice-work like structure, the crosslinks of which create “pores”. The ratio of linear polymer to crosslinkers is important for determining pore size, so make sure you are consistently using and ORDERING the correct mix. Also, VERY IMPORTANT! Unpolymerized acrylamide is a neurotoxin: Be safe, and don’t forget to tell the undergrad.
  • Tris. Tris is the buffer of choice to regulate gel’s pH. Stacking buffer is usually pH 6.8 and resolving gel is pH 8.8.
  • 10% SDS. SDS is the negatively charged detergent that will coat your proteins so that they resolve based on mass. Some commercial gels don’t even bother to put it inside the gel, but most who pour their own gels do. It just seems prudent, don’t you think?
  • 10% ammonium persulfate (APS). APS initiates the polymerization of your acrylamide mix by providing a free radical necessary for polymerization to take place.
  • TEMED. This is the catalyst (and yes, it does reek like rotting fish). Your polymerization reaction would still take place without this molecule but it would takes hours, but if you add (and you should!) TEMED it happens in minutes. So once you add the TEMED don’t waste anytime pouring your gel solution.

Gel Electrophoresis

It is important to remember that pH in gel electrophoresis is critical. If your pH is off, the stacking gel cannot properly prepare your proteins for a coordinated entry into the resolving gel. Under proper pH conditions, however, when a current is applied “leading and trailing ions” (of chloride and glycine, respectively) will be created in the running buffer. These ions work to sandwich and compact your proteins samples in the stacking gel ensuring that they enter the resolving gel evenly. Once the proteins exit the stacking and enter the resolving gel, the pH changes, freeing your proteins to traverse the resolving gel at a speed relative to their mass.

Transferring

Okay, so while the concept of using electricity to transfer your proteins from your gel to a membrane is straightforward, some details are often overlooked. Including the important detail of how the proteins stick to the membrane once there. Hint: It is not a covalent interaction! Instead, it is a very strong “sticky” force involving both hydrophobicity and electrostatic charges between the protein and the PDVF or nitrocellulose membrane. So why is this forgettable detail important? Because, this chemistry dictates how well your protein-of-interest sticks and if/when it will unstick. As your best blot will always be the first probing, it is best to always probe for your weakest antibody first, as subsequent handling and stripping can unstick your proteins-of-interest.

Blocking the membrane

Before you start throwing antibody on your membrane, you need to prep it by coating all the unoccupied sticky portions of the membrane with a blocking agent. There are a number of blocking agents to choose from, and even some buffers such as Tris supposedly stick to the membrane to facilitate the block. The two most commonly used blocking agents are milk and bovine serum albumin (BSA). While milk is the old standby, some antibodies nonspecifically stick to the milk, which causes dirty blots and in the process effectively dilute your antibody. Also, this “dirty” milk can contain phosphatases that can in theory remove your phosphorylation groups (or other post-translational events). Thus, because it has been through some purification steps, BSA is the preferred blocking agent when probing with phosphorylation sensitive antibodies. Pay attention if you decide to use BSA, as there are different levels of purified BSA with very different price tags. There is also a barnyard animal clause in blocking choice. If your primary antibody was made against a barnyard animal (cow, horse, goat, donkey) then you can’t use cow proteins (like the BSA or milk) to block due to cross-reaction or IgG contamination. Instead, you will need to block with something like chicken, rabbit, or undergrad serum (A joke!! Don’t sue me if you hurt the undergrad).

The Primary Antibody

Your primary antibody of choice can either be an esoteric biological homing device or a waste of $350. And you never know which until you try it! Just remember the basic concepts of antibody recognition: Antibodies are specialized immune proteins that recognize and bind to specific protein sequences/structures called epitopes. And if you are lucky these epitopes are on your protein-of-interest! To increase your luck, remember that epitopes are often species-specific and that your antibodies/protein interaction may be sensitive to denaturing conditions and post-translation modifications.

The Secondary Antibody

Secondary antibody selection depends on your primary antibody and your detection system. Secondary antibodies recognize the heavy (constant) chain of your primary antibody and this is species-specific. So pay attention to what species your primary antibody was made in! And what antibodies might be in your sample! As would be the case if you did immunoprecipitation. Luckily, some special secondary antibodies only recognize disulfide-bonded primary antibodies, thereby eliminating the chance of seeing antibodies used previously in the experiment.

Secondary antibodies are conjugated to different developing molecules depending on the type of developing system you are using. For film you will likely use HRP (horseradish peroxidase) or AP (alkaline phosphatase); for laser capture, you will need a fluorophore.

Developing the Blot

How you develop your Western blot depends on your developing system. In other words, what developing molecule is coupled to the heavy chain of your antibody (or in some short-cut cases, your primary antibody). For example: If the HRP is conjugated to your secondary antibody, your membrane needs to be incubated with a luminol-containing kit. The resulting chemiluminescence can then be captured on film in a dark room, or with a camera image detector. If, however, your secondary was conjugated to fluorophores, you can use a fluorescent scanning device and scan immediately. Very nice! These systems are also nice because they can allow you to probe multiple antibodies simultaneously.

Want all your Western blot buffer recipes, an ECL reagent recipe, and an easy protocol all in one place and on hand? Download our free Western blot cheat sheet.

Feeling good about your knowledge of Western blot basics and jargon now? Ready to learn more? Great! See my other article about more advanced Western blotting topics here.

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