Quantifying Protein with Indirect Immunostaining

Recently, I’ve been working as a research assistant in a lab and finally came across the technique of Western blot. I didn’t have any training on this technique because I didn’t take molecular biology lab in my undergraduate career, so I had to learn it on the spot.

I decided to write this blog post as a way to document what I’ve learned so far. I will be going through the immunostaining procedure, including blocking, washing, blotting, primary and secondary antibody incubation, and detection.

A Brief Introduction

When it comes to detecting specific proteins in a mixture of many kinds of protein, for example, a sample obtained from a tissue extract, gel electrophoresis (most commonly SDS-PAGE) followed by antibody is a common approach.

The proteins are first separated in electrophoresis by size, and then transferred to a solid membrane. The entire process, known as Western blotting, allows for the detection and relative quantification of specific proteins using specific antibodies.

Immunostaining is based on the specific binding of primary antibodies to their target molecules, which are then visualized through the use of labeled secondary antibodies. By using the appropriate antibodies and detection methods, we can learn about protein expression patterns, cellular localization, or signaling pathways, among other aspects of cellular function.

Indirect vs. Direct Immunostaining

You may notice the word “indirect” in my blog post title. This is because there are two main variants of antibody staining: indirect and direct.

Direct immunostaining uses a single primary antibody that specifically binds to the target molecules. This primary antibody is labeled and can be directly conjugated to a detection molecule, such as a fluorochrome or an enzyme, that allows for visualization of the target molecule.

On the other hand, indirect immunostaining uses an unlabeled primary antibody that binds to the target of interest, followed by a secondary antibody that is labeled with a detection molecule. Here, the secondary antibody binds specifically to the primary antibody. In this case, the secondary antibody amplifies the signal by binding to multiple sites on the primary antibody, increasing the sensitivity of the staining.

So the key difference here is the use of either a single labeled primary antibody (direct) or an unlabeled primary antibody and a labeled secondary antibody (indirect) to detect the target molecule.

Monoclonal vs. Polyclonal Antibodies

When choosing an antibody, you may also see the term monoclonal or polyclonal. In short, these are two types of antibodies produced by different B cells and have different properties.

Monoclonal antibodies are antibodies derived from a single clone of B cells that all produce identical antibodies. Here, a single clone of B cells is selected and grown in culture to produce large quantities of identical antibodies that all recognize the same epitope (a specific part of an antigen) on the target molecule.

Polyclonal antibodies, on the other hand, are produced by multiple clones of B cells that produce different antibodies against different epitopes of a target molecule. These antibodies are less specific than monoclonal antibodies, because they recognize multiple epitopes on a target molecule.

The choice between monoclonal and polyclonal depends on your specific needs. Using a monoclonal antibody can not only provide a more consistent signal, but also be used to detect post-translational modifications or protein-protein interaction. In contrary, polyclonal antibodies can provide a broader recognition of the target protein.

Blotting: From Gel to Membrane

After separating a mixture of proteins in gel electrophoresis, the proteins need to be transferred to a solid support. Typically we use a membrane for that purpose. This process is called a electrophoretic transfer, or simply blotting. The transfer is achieved by placing the membrane between the gel surface and the positive electrode, while applying an electric field perpendicular to the surface of the gel. The proteins then move out of the gel and onto the membrane, because the negatively charged proteins are attracted to the positively charged electrode.

Blocking: Avoiding Non-specific Binding

As the term suggests, blocking involves blocking the remaining binding surface on the membrane to prevent non-specific binding of antibodies. This process is done after the transfer of proteins onto the membrane.

Antibodies can bind non-specifically to the membrane, which can generate a lot of background noise and can compromise the accuracy of our results. Therefore, by blocking the remaining binding sites, we create a uniform and neutral surface on the membrane, which the antibody cannot bind to. It improves the specificity of antibody binding and enhances the signal-to-noise ratio when we want to visualize the membrane.

Washing: Removing Unbound Molecules

It is also essential to include washing step. Washing the membrane allows for the elimination unbound reagents and helps minimize background noise. If the washing step is insufficient, you may see high background signals when visualizing the proteins. On the other hand, excessive washing may cause decreased sensitivity due to the elution of the antibody from the blot.

Antibody Incubation

After blocking and washing the membrane, we can finally begin antibody incubation. As mentioned above, the procedure of indirect immunostaining involves the utilization of two antibodies. Choosing the right antibodies for indirect immunostaining depends on many factors, such as the type of antigen, the host species, the required specificity, and the available detection methods.

To begin with, it is extremely important to select an antibody that recognizes the specific antigen of interest. This will be different for each study, and you will have to do the hard work of conducting literature research to find out which would be suitable for your interest.

At the same time, it is important to consider the sample being analyzed because we want to reduce background noise and avoid cross-reactivity between the primary antibody and the sample. For example, if you are studying a mouse protein, it’s recommended to select a primary antibody raised in a different species to avoid potential cross-reactivity. Here, a primary antibody produced in rabbit can be a suitable option, which can be detected with an anti-rabbit IgG secondary antibody.

Moreover, the specificity of the antibody should be considered. Here, we are referring to the above-mentioned monoclonal and polyclonal antibodies.

Finally, the available detection methods should also be taken into consideration. The two major types of detection methods include chemiluminescence and fluorescence. You should choose the antibody that works best with the equipment you are using.

Indirect antibody staining, as mentioned above, requires two antibodies. It is important to wash the membrane between the primary and secondary staining process.

Detection Methods

After you have stained the membrane with your primary and secondary antibody, it’s time to visualize the proteins. This process is called detection.

When it comes to detection, chemiluminescent and fluorescence are the two most commonly used methods.

In chemiluminescent detection, the antibodies are labeled with an enzyme that reacts with a substrate to produce light. Often, the enzyme HRP, or horseradish peroxidase, is used in this method. The presence of the target protein is made visible by using a chromogenic substrate that, when oxidized with H2O2 as an agent, yields a luminescent or a color change. This change can be detected by spectrophotometric methods. Here, the intensity of the light correlates with the amount of the target protein present. In our lab, we use the ChemiDoc Imaging System for that purpose.

On the other hand, fluorescence detection uses fluorescent tags attached to the antibodies that bind to the target protein. When excited with a specific wavelength of light, these tags emit light of a different wavelength that can be visualized with a specialized camera. We use our LI-COR imaging systems.

Stripping: Reuse the Membrane

Stripping is a method commonly used in which the primary and secondary antibodies are removed from the membrane. After stripping, the membrane can be re-probed with a different set of antibodies to visualize different proteins or optimize detection of a protein. This technique saves time and resources by eliminating the need for multiple gels and transfers.

The stripping buffer usually contains SDS, Tween-20, and Glycine. HCl are also added to lower the pH to 2.2. The low pH of the stripping buffer removes bound antibody by altering their structure, so their binding sites are no longer active.

Final Words

In this post, I discussed several steps that follow protein gel electrophoresis, such as blocking, washing, staining, detection, and stripping. As I had no prior experience, it is essential for me to understand the underlying mechanisms behind each step to ensure that I follow the protocol correctly and avoid any errors. It will need some practice to make sure I choose the correct primary and secondary antibodies, but I do hope in the future I can perform these procedures confidently and achieve reliable results.


  • Mahmood T, Yang PC. Western blot: technique, theory, and trouble shooting. N Am J Med Sci. 2012 Sep;4(9):429-34. doi: 10.4103/1947-2714.100998. PMID: 23050259; PMCID: PMC3456489.




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