Unveiling the Western Blot Mystery: Do Multiple Bands Signal a Specificity Crisis for Antibodies?

Western blotting is a widely used technique in molecular biology, biochemistry, and immunogenetics for detecting and analyzing the expression of specific proteins in biological samples.

At the beginning of the experiment, a mixture of proteins is separated by molecular weight using polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins are then transferred onto a solid support membrane, such as nitrocellulose or PVDF, via electrotransfer, forming a stable antigen–carrier complex.

Next, the membrane is incubated with a primary antibody (1° Ab) that specifically binds to a defined epitope on the target protein. This step ensures high specificity, as the primary antibody recognizes and binds only to the protein of interest.

After primary antibody incubation, a secondary antibody (2° Ab) is added. The secondary antibody is designed to bind to the Fc region of the primary antibody and is typically conjugated to a detectable label, such as an enzyme or fluorophore. This binding amplifies the signal, enhancing the sensitivity of the detection system.

Finally, through the application of appropriate substrates—such as chemiluminescent or fluorescent reagents—the signal is visualized, allowing for the qualitative and semi-quantitative analysis of the target protein on the membrane (see Figure 1).

Figure 1. Western Blot Analysis Flowchart

A key step in the Western blot assay is the use of a specific primary antibody (“primary Ab”) that binds to the antigenic epitope on the target protein. The choice of primary antibody is critical to the accuracy of the results. In theory, antibodies recognize their target antigens through specific binding sites, enabling selective detection (Xu et al., 2022).

So, why do multiple bands sometimes appear in a Western blot analysis? Does this indicate poor antibody specificity?

In fact, the presence of multiple bands is not necessarily a direct consequence of low antibody specificity. Rather, it may result from a variety of complex factors. In addition to antibody specificity, several other common reasons include:

1. Post-Translational Modifications (PTMs)

Many proteins undergo various post-translational modifications such as phosphorylation, glycosylation, and ubiquitination, which can alter their electrophoretic mobility, leading to the appearance of multiple bands on the gel (Xu et al., 2023).

For example, CD147 (Cluster of Differentiation 147), a highly glycosylated transmembrane protein with a theoretical molecular weight of 42 kDa, often appears as multiple bands ranging from 38 to 70 kDa in Western blotting due to different glycosylation states (Figure 2).

Figure 2. Glycosylation Modifications Lead to Multiple Bands of CD147 in Western Blotting

2. Proteolytic Processing

Many proteins undergo specific cleavage or processing events to become biologically active after translation.

For instance, Caspase-9, a key enzyme in the apoptosis pathway, exists as an inactive precursor (~47 kDa) in normal human cells. Upon apoptotic signaling, the precursor is cleaved at the Asp315-Asn316 site into ~37 kDa N-terminal and ~35 kDa C-terminal fragments. These fragments then non-covalently assemble into active homo- or heterodimers, activating downstream caspases such as Caspase-3, -6, and -7.

Thus, antibodies against Caspase-9 may detect not only the full-length precursor but also its cleaved fragments in Western blotting (Figure 3).

Figure 3.Caspase-9 Antibody Recognizes Both Full-Length Precursor and Cleaved Fragments of Caspase-9

3. Presence of Multiple Isoforms

Alternative splicing, post-translational modifications, or gene duplications may lead to multiple isoforms of a target protein with different structures or sizes. Although these isoforms may differ functionally or structurally, they often share immunogenic epitopes and can be recognized by the same antibody (Blijdorp et al., 2021).

For example, ERK1 (MAPK3, ~44 kDa) and ERK2 (MAPK1, ~42 kDa) are two highly homologous isoforms of extracellular signal-regulated kinases (ERKs), part of the mitogen-activated protein kinase (MAPK) family. A general anti-ERK antibody may detect both isoforms, resulting in two distinct bands on the Western blot (Figure 4).

Figure 4. ERK Antibody Recognizes Both ERK1 and ERK2 Isoforms

4. Protein Degradation

Protein homeostasis in cells is a dynamic process that maintains a balance between protein synthesis and degradation to preserve cellular function. Antibodies may recognize both full-length proteins and degradation products, resulting in multiple bands (Rahman and Sadygov, 2017).

Beyond biological factors, technical variables during the Western blot procedure—such as gel preparation, electrophoresis conditions, transfer efficiency, blocking, and antibody incubation—must be rigorously controlled. Any error or inconsistency can introduce non-specific bands or background noise.

So, when multiple bands appear in a Western blot experiment, how can we determine whether they result from intrinsic properties of the protein or from poor antibody specificity?

First, it is essential to define specificity. Specificity refers to an antibody’s ability to bind only to its intended antigen and not to other unrelated proteins (Pillai-Kastoori et al., 2020). In other words, a specific antibody should recognize and bind only to its target protein, without cross-reacting with others.

Antibody specificity is one of the most important criteria in evaluating antibody quality. There are various methods to validate antibody specificity, and among them, genetic approaches are globally recognized as the most reliable and direct. These include: Gene knockout (KO) at the DNA level, and Gene knockdown (KD) at the mRNA level using small interfering RNA (siRNA) or short hairpin RNA (shRNA).

If an antibody yields a clear signal in wild-type (WT) cell lysates, but the signal is significantly reduced or disappears in KO or KD lysates, the antibody is considered to have high specificity.

As shown in Figure 5, antibodies producing multiple bands were validated using shRNA-mediated gene silencing. The results clearly demonstrate that these so-called “non-specific” bands were, in fact, specific products of the target protein. In other words, “extra bands” are not necessarily “nonspecific”.

Figure 5. Validation of Multiple-Band Antibodies by shRNA-Mediated Gene Silencing

A: Western blotting analysis of CD147 protein expression in wild-type (WT) and CD147 knockdown (KD) HeLa cells.

B: Western blotting analysis of Caspase-9 protein expression in wild-type (WT) and CASP9 knockdown (KD) HeLa cells.

C: Western blotting analysis of ERK protein expression in wild-type (WT) and MAPK3/MAPK1 knockdown (KD) HeLa cells.

In conclusion, the appearance of multiple bands in Western blotting does not necessarily indicate poor antibody specificity. The most effective way to validate antibody specificity is through gene knockdown or knockout, which results in the disappearance of the target protein encoded by the corresponding gene.

Our company has developed a series of validated lentiviral reagents and a catalog of high-specificity antibodies through our proprietary ShGE™ gene knockdown platform. These biological tools offer reliable resources and services for basic biomedical research and CRO-based clinical studies.

References：

1. Blijdorp, C.J., Tutakhel, O.A.Z., Hartjes, T.A., van den Bosch, T.P.P., van Heugten, M.H., Rigalli, J.P., Willemsen, R., Musterd-Bhaggoe, U.M., Barros, E.R., Carles-Fontana, R., et al. (2021). Comparing Approaches to Normalize, Quantify, and Characterize Urinary Extracellular Vesicles. J Am Soc Nephrol 32, 1210-1226. 10.1681/asn.2020081142.

2. Pillai-Kastoori, L., Heaton, S., Shiflett, S.D., Roberts, A.C., Solache, A., and Schutz-Geschwender, A.R. (2020). Antibody validation for Western blot: By the user, for the user. J Biol Chem 295, 926-939. 10.1074/jbc.RA119.010472.

3. Rahman, M., and Sadygov, R.G. (2017). Predicting the protein half-life in tissue from its cellular properties. PLoS One 12, e0180428. 10.1371/journal.pone.0180428.

4. Xu, L., Abd El-Aty, A.M., Shim, J.H., Eun, J.B., Lei, X., Zhao, J., Zhang, X., Cui, X., She, Y., Jin, F., et al. (2022). Design and Characterization of a Novel Hapten and Preparation of Monoclonal Antibody for Detecting Atrazine. Foods 11. 10.3390/foods11121726.

5. Xu, Q., Wang, Y., Chen, Z., Yue, Y., Huang, H., Wu, B., Liu, Y., Zhou, D.X., and Zhao, Y. (2023). ROS-stimulated protein lysine acetylation is required for crown root development in rice. J Adv Res 48, 33-46. 10.1016/j.jare.2022.07.010.

08/04/2025

Ming Xv

Director of Production

GenuIN Biotechnologies