Loading Control Antibody Guide
Knockout-validated primary antibodies
Secondary antibodies resources
Alexa Fluor secondary antibodies
Biotinylated secondary antibodies
Gold-labelled secondary antibodies
How to prepare Phosphate Buffered Saline (PBS)
How to design a flow cytometry panel
Enhancing Detection of Low-Abundance Proteins
9 tips for detecting phosphorylation events using a Western Blot
Western Blotting with Tissue Lysates
Western blot membrane stripping protocol
Immunohistochemistry and Immunocytochemistry
Chromogenic and Fluorescent detection
Preparing paraffin-embedded and frozen samples for Immunohistochemistry
Competitive ELISA assay protocol
Measuring analyte concentration using serial dilutions and standard curve
What is immunodetection?
Shortly after blotting, the matching antibodies can detect the protein of interest. This procedure consists of several steps:
Blocking: the blot with the transferred protein is incubated with either a detergent or a protein covering the whole surface in order for the antibodies not to bind non-specifically to the membrane.
Incubation of the antibody: the matching antibody binds to the protein of interest.
Using a substrate to detect the target protein: the antibody has a label, usually an enzyme like HrP that can be detected via a substrate. That substrate produces a visual signal that corresponds to the target protein’s position.
This is the most crucial step of Western blotting, because if done correctly, it can prevent the non-specific binding of the antibody to the membrane. Usually, blocking solutions have between 3 to 5% BSA or non-fat dried milk in a solution of phosphate buffered saline.
To better help the process, a small amount of detergent solution is also incorporated in both the blocking and washing solutions in order to reduce the background staining. Furthermore, non-fat dried milk is a good choice because it is both cost-effective and has many uses.
It is important to notice that because milk contains casein and biotin, it has the tendency to interfere with assay results. When this step is over, the blot must be washed and incubated in a diluted antibody solution.
After being placed in a diluted antibody solution, the blot is incubated either for several hours at room temperature or overnight at 4C. When preparing the diluted solution, the general rule is that purified antibodies are diluted down to a 1-10 mg per ml, but it is best to check the recommendations for the product or to contact the manufacturer for more information.
Make sure that during all these steps adequate agitation and volumes are present, so the blot is evenly exposed, and no drying occurs.
Direct and Indirect detection:
The two different ways of detection both have their advantages and limitations. Direct detection is a one-step procedure, compared to the indirect, which has two steps.
Direct detection offers less chance of non-specific signal and is faster, but labelling to the primary antibody can affect the antibody’s ability to bind to the target protein and furthermore labelling every primary antibody is more expensive and takes more time.
Indirect detection has stronger signal, mainly because several secondary antibodies bind to a single primary one and changing labels is as easy as switching the secondary antibodies. Because of these qualities, then the primary antibodies are from the same species a significant amount of time and money can be saved and more different labels are available.
On the other hand, if the secondary antibody binds to other proteins on the blot the specific signal can significantly decrease and more incubation and washing is needed, which respectively adds more time to the experiment.
Detection with substrate:
After the target protein has been tagged with a labelled antibody, and the leftover antibody has been washed away, the label can be used to identify the location of the target protein on the blot.
Some labels can be detected instantly, without any additional processing. Fluorescent tags, for example, only require the right equipment to observe and record the fluorescent signal.
Usually, HrP, a small, stable enzyme with high specificity and rapid turnover is used in Western blots. It is deactivated by sodium azide, so it is imperative that no azide is present in the blocking, dilution, or washing solutions.
The HrP label is detected after exposing it to a substrate solution in the last step of the immunodetection procedure. Substrate solutions for Western blotting are chemical reagents that are acted upon by the enzyme in order to obtain a signal that can be easily measured. HrP label is detected with either colourimetric or chemiluminescent substrates.
Colourimetric substrates for HrP create brown or purple/black bands on the surface of the blot. These substrates are simple and straightforward to use and produce visible bands in several minutes up to a couple of hours. The detection limits for them are in the low nanogram range.
Types of blots:
Test blots are the simplest type of Western blots that are used for the sole purpose of optimizing or troubleshooting experimental conditions. They are often produced by running several lanes of the same lysate or purified protein solution on a gel, and after the transfer, the blot is cut into strips, so it can be tested individually.
They provide a fast and efficient way of examining different antibody dilutions or detection substrates. Dot blots and slot blots are a quite useful variation on the traditional Western blot. They skip the gel electrophoresis, so there is no separation of proteins by size, so the target protein or cell lysate mixture is added directly onto the surface of the membrane.
Protein solutions can be applied directly in a small volume, to produce a neat grid of samples. Each blot contains known amounts of target protein or cell lysate. After they have dried, the blots are subjected to the same steps used for traditional Western blotting.