Western Blotting

Preface: I would like to start a column in which anyone can share their experiences with any laboratory technique. Briefly, after the introduction and explanation of the technique, some tips, results, files and troubleshooting useful for anyone are shared. So what follows is the first draft which will be modified from time to time to improve its quality.

Utility: Western blot is a technique used for protein analysis that allows the identification and quantitation of specific proteins in complex protein mixtures derived from cell or tissue samples. This method is widely used because it is relatively cheap and straightforward compared to other methods.

Description: The Western blot process is divided into four main phases: cell lysis and sample preparation, electrophoretic separation of proteins based on size, transfer of proteins to a stable membrane, and visualization of the proteins of interest using specific antibodies. These phases are outlined below:

Phase 1: Cell Lysis and Sample Preparation: You start with a lysate sample, which is a mixture of a bunch of proteins that have been released from cells. You accomplish this by use of a detergent that breaks down the cell membranes, so now the proteins spill out, and all get mixed together. The lysate is then mixed with a reagent that cleaves disulfide bridges, the links proteins often times have between chains that help give them structure. At the same time you also mix it with a detergent (usually SDS) that linearizes the protein. That is to say, the proteins completely unravel. Instead of a bunch of complex shapes, you now have a bunch of strings. The strings are of various lengths, since the starting proteins were a variety of sizes. The initial structure of the proteins is now taken out of the equation. The detergent also coats them with a uniform negative charge. This is important.

Phase 2: Electrophoretic Separation: The now linearized and negatively charged proteins are loaded into the top of a gel, and a current is run through the gel such that the bottom of the gel has a positive charge. Proteins negative, bottom of gel positive, so the current pulls the proteins through the gel. The gel is a mesh which the proteins have trouble moving through. Shorter proteins can move more easily through it, while longer ones have more difficulty. Since the shape of the proteins is no longer a factor, as they’ve been linearized, the only determining factor for the mobility of proteins is their length. Longer protein = more amino acids = more mass. This separation allows for the isolation and identification of specific proteins from complex mixtures.

Phase 3: Protein Transfer: the separated proteins are transferred to a support membrane, where they bind and can be further analyzed. This transfer is commonly achieved through electrophoretic transfer. There are several transfer techniques:

Tank transfer: offers the widest range of settings for both voltage and transfer times.
Semi-Dry: the gel and membranes are placed between two layers of filter paper soaked with buffer and placed in direct contact with flat electrodes. Slightly easier to set up compared to tank systems. There are some limitations on voltages and transfer times (15-60 minutes). These systems are ideal for molecules in the 30-120 kDa range.
Rapid transfer systems: offer faster transfer times (3-10 minutes) and use pre-soaked filter papers and membranes prepared in disposable packaging, so manual setup is practically nil.

Phase 4: Protein Detection: This membrane is then immersed in a liquid containing an antibody which binds to your protein of interest. This antibody can have a light-producing enzyme on it, or it can require the binding of a second antibody (called a secondary antibody) which has the light producing enzyme. At the end of it, you end up with a glowing band where your protein is. Antibodies aren’t perfect, so knowing where to look helps us avoid accidentally quantifying the wrong band. It's important to first block your membrane, before adding your antibody. Blocking non-specific sites on the membrane is crucial to prevent non-specific binding of antibodies, thus ensuring clear and readable results. Various blocking agents can be used for this purpose, including bovine serum albumin (BSA) and skim milk powder, although the choice of blocking agent depends on compatibility with the chosen detection method.

How to Interpret: More protein = bigger, darker band. Western blots are often done with housekeeping genes, which are proteins that should be present at the same level in all samples. You normalize the signal of your protein of interest relative to these. They help account for inconsistency in loading the gel. If I load half as much of one sample, I’ll see half as much of my protein, but also half as much of my housekeeping gene. Blots for housekeeping genes are usually shown at the bottom, such as GAPDH or Beta-Actin.
You can be semi-quantitative with western blotting by using something called densitometry, which is a way to analyze an image and assign a numerical value to how intense the band signal is. This is a totally legitimate practice, but must be performed carefully. The signal of the bands is imaged with a CCD camera, which has a set dynamic range. At a certain point, a signal can be so bright that it tops out the sensor. You could have two samples where one is much brighter than the other, but if they’re both so bright they top out the sensor, so they would both appear the same.

Example of a western blot result:

Assay Tips:

Never leave a well empty. If you don't have samples to load, simply use buffer with water or RIPA, as proteins might migrate differently than expected if it were empty.
Use low voltage to separate close molecular weight bands and improve the transfer efficiency for large proteins.
Before loading samples into the wells, pipette to remove bubbles.
For phosphorylated proteins, it's preferable to use BSA as a blocking agent.
If, once you develop the Western, you get a black band with a white band inside, it's likely that you've saturated the signal. So, you've incubated the membrane with too strong a substrate or you need to dilute the primary or secondary antibody more.
When preparing samples, prepare a greater volume as the sample may evaporate during denaturation.
Ensure you are using validated antibodies for your protein of interest
When analyzing multiple proteins per blot, make sure they are either resolved from each other in terms of mass, or you're using a different species of primary antibody for each to minimize signal overlap

How to load the samples: https://docs.google.com/spreadsheets/d/17dg9bGnWT_FfJHTid-nkXE_iRbLf_OEnB2AYxjqlhZA/edit?usp=sharing

Preprint

Preprint

Western Blotting

Western Blotting