Western blot (sometimes called the protein immunoblot) is a widely used analytical technique used in molecular biology, immunogenetics and other molecular biology disciplines to detect specific proteins in a sample of tissue homogenate or extract. The typical western blot protocol that Abbkine antibodies are subjected to is reproduced below.
Note: Some of the steps in this protocol require optimization depending on the sample and antibody being used.
1. Sample preparation
Protein can be extracted from different kind of samples, such as tissue or cells. Lysis buffers differ in their ability to solubilize proteins, with those containing sodium dodecyl sulfate (SDS) and other ionic detergents considered to be the harshest and therefore most likely to give the highest yield.
As soon as lysis occurs, proteolysis, dephosphorylation and denaturation begin. These events can be slowed down significantly if samples are kept on ice or at 4°C at all times and appropriate inhibitors are added fresh to the lysis buffer. To denature, use a loading buffer with the anionic detergent sodium dodecyl sulfate (SDS), and boil the mixture at 95–100°C for 5 min. Heating at 70°C for 5–10 min is also acceptable and may be preferable when studying multi-pass membrane proteins. These tend to aggregate when boiled and the aggregates may not enter the gel efficiently.
The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pI), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. This is a very useful way to identify a protein.
By far the most common type of gel electrophoresis employs polyacrylamide gels and buffers loaded with sodium dodecyl sulfate (SDS). SDS-PAGE (SDS polyacrylamide gel electrophoresis) maintains polypeptides in a denatured state once they have been treated with strong reducing agents to remove secondary and tertiary structure (e.g. disulfide bonds [S-S] to sulfhydryl groups [SH and SH]) and thus allows separation of proteins by their molecular mass. Sampled proteins become covered in the negatively charged SDS, effectively becoming anionic, and migrate towards the positively charged (higher voltage) anode (usually having a red wire) through the acrylamide mesh of the gel. Smaller proteins migrate faster through this mesh, and the proteins are thus separated according to size (usually measured in kilodaltons, kDa). The concentration of acrylamide determines the resolution of the gel – the greater the acrylamide concentration, the better the resolution of lower molecular weight proteins. Typically, 10–50 µg of protein is loaded per well depending on protein abundance, molecular weight, and type of gel used.
Figure 1 The gel percentage required for the size of proteins
To make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The primary method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. An older method of transfer involves placing a membrane on top of the gel, and a stack of filter papers on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. In practice this method is not used as it takes too much time; electroblotting is preferred, in which case, as with PAGE-SDS, proteins migrate toward the (+) anode (red wire on most instruments). As a result of either “blotting” process, the proteins are exposed on a thin surface layer for detection (see below). Both varieties of membrane are chosen for their non-specific protein binding properties (i.e. binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand up well to repeated probings.
Figure 2 Western blot transfer
Since the membrane has been chosen for its ability to bind protein and as both antibodies and the target are proteins, steps must be taken to prevent the interactions between the membrane and the antibody used for detection of the target protein. Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein – typically 3–5% bovine serum albumin (BSA) or non-fat dry milk (both are inexpensive) in tris-buffered saline (TBS) or I-Block, with a minute percentage (0.1%) of detergent such as Tween 20 or Triton X-100. Although non-fat dry milk is preferred due to its availability, an appropriate blocking solution is needed as not all proteins in milk are compatible with all the detection bands. The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached. Thus, when the antibody is added, there is no room on the membrane for it to attach other than on the binding sites of the specific target protein. This reduces background in the final product of the western blot, leading to clearer results, and eliminates false positives.
5. Probing with primary and secondary antibodies
During the detection process the membrane is “probed” for the protein of interest with a modified antibody which is linked to a reporter enzyme; when exposed to an appropriate substrate, this enzyme drives a colorimetric reaction and produces a color. For a variety of reasons, this traditionally takes place in a two-step process, although there are now one-step detection methods available for certain applications. The amount of primary and secondary antibody will vary depending on the antibody being used and the samples you are running. A good starting point is to check the antibody product pages for recommended dilution ranges and from there titrate to find the optimal antibody dilution for the experiment.
6. Developing the blot
After the unbound probes are washed away, the western blot is ready for detection of the probes that are labeled and bound to the protein of interest. In practical terms, not all westerns reveal protein only at one band in a membrane. Size approximations are taken by comparing the stained bands to that of the marker or ladder loaded during electrophoresis. The process is commonly repeated for a structural protein, such as actin or tubulin, that should not change between samples. The amount of target protein is normalized to the structural protein to control between groups. A superior strategy is the normalization to the total protein visualized with trichloroethanol or epicocconone. This practice ensures correction for the amount of total protein on the membrane in case of errors or incomplete transfers.
HRP western blot
Develop the blot using ECL Chemiluminescent Substrate (K22020 SuperLumia ECL HRP Substrate Kit) for 1min. Place the membrane in clear film and remove any excess substrate. If the exposure time is longer than a few min, consider using a more sensitive substrate, such as (K22030 SuperLumia ECL Plus HRP Substrate Kit).
Fluorescent western blot
Acquire image using a fluorescent imager. Place the blot on the imaging surface and ensure it is lying flat with no air bubbles. If you are multiplexing, use the 800 nm channel for detection of less abundant proteins or weak targets. Use the 680 nm channel for detection of more abundant proteins or strong targets.