How to achieve greater separation in Western blot when proteins have a similar size

Despite its overall simplicity, the Western Blot application is an effective procedure for the immunodetection of proteins and an essential tool within biological research. Nevertheless, proteins of the same length or similar size, such as isoforms of the same protein, are difficult to separate. With some adjustments, you can however achieve great results. Here we share some tips.

The Western Blot (WB) application is one of the most frequently antibody-based methods used in many fields of biochemical research. It is a simple way of detecting the presence or absence of a particular protein in a complex mixture, to compare protein levels, protein abundance, kinase activity, cellular localization, protein-protein interactions, or monitoring of post-translational modifications in different conditions or different tissues. If appropriately undertaken, WB is reliable, quantitative, both in relative and absolute terms, and extremely valuable (Bass et al., 2017).

WB combines the superior resolving power of polyacrylamide gel electrophoresis (PAGE) the specificity of antibodies, and the sensitivity of enzyme assays. The first step in WB is to prepare the protein sample by mixing it with the detergent called sodium dodecyl sulfate (SDS), which makes the proteins unfold into linear chains and coats them with a negative charge. Next, the protein molecules are separated according to their sizes using PAGE. Following separation, the proteins are transferred from the gel onto a blotting membrane, usually, a nitrocellulose membrane, which binds and immobilizes the proteins followed by antigen detection by primary antibodies specific for the protein of interest, incubation with a secondary antibody linked to chemiluminescent or a fluorescent tag or label, develop, detection and quantification.


How to achieve greater separation of proteins of similar size?

One limitation of WB is that proteins of the same length or similar size are difficult or even impossible to separate.

Imagine working with a protein that comes as two in vivo translation products: a full-length protein and a truncated form, differing by just a few kDa in their molecular weight: you aim to obtain the highest possible degree of separation of these two protein variants while, at the same time, ascertain the high resolution of each band.

Some post-translational modifications such as glycosylation and SUMOylation can cause a huge change in the molecular weight of a protein. Other, such as phosphorylation, causes only minimal changes in molecular weight. The common procedure in the latter case is to use a phospho-specific antibody to detect the phosphorylated form of the protein first, then strip the membrane and apply a pan-antibody specific for the total form of the protein. This procedure, however, comes with several inherent difficulties and cons, such as increased background, and lack of reproducibility and it doesn’t really save time. So, there must be another way.


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How to separate proteins of similar size? 

How to separate proteins of similar size? Here are some tips:

  • In WB, proteins are separated by their molecular weight. The larger the proteins, the less and the slower they travel within the gel and vice versa. Proteins can travel faster through a gel that contains a lower percentage of polyacrylamide. Hence a low-density polyacrylamide gel would be able to separate proteins that are close in molecular weight with higher resolution. Generally, lower molecular weight proteins are best resolved on high percentage gels, whereas larger proteins require lower percentage gels for optimal resolution. For example, for proteins of <10 kDa use 15% SDS/PAGE gel, for 10-30 kDa 12%, for 30-100 kDa 10%, and for >100 kDa use 8%.
  • Gradient gels are the ideal gel to use during a WB, especially for an experiment focused on the separations of proteins of similar molecular weight, only if the difference in the protein size is not too small (ideally it should be >10 KDa). Gel percentage directly correlates to protein size and resolution, so choosing the correct gradient is the key to a well-resolved gel. Gradient or discontinuous SDS/PAGE gels are made on the resolving gel so that there is a gradual dilution from a high to low concentration from the bottom region of the gel to the top like 3-8%, 8-25%, and 4-12%.
  • Multiplexed fluorescence WB is another way to visualize protein that differs in size only by a few kDa by detecting two epitopes on a single protein. Using fluorescent tags and monoclonal antibodies of different isotypes permits the detection of multiple bands on the same gels. This multiplex fluorescent method is very useful to analyze changes in target protein phosphorylation, glycosylation, acetylation, ubiquitination, and other post-translational modifications.
  • The two-dimensional (2-D) immunoblotting involving the separation of proteins in two dimensions (according to the isoelectric point in the first dimension and according to the molecular weight in the second dimension) might also help. Clearly, 2-D gel electrophoresis will help only if the two isoforms have different isoelectric points.
  • For a better resolution of proteins with similar sizes, another solution is to choose a long-length gel or run the gel for an extra time until you reach the desired resolution.


Figure 1.  Schematic of proteins of similar size resolved by electrophoresis in different gel running systems.


“The molecular weight of my protein is known, and therefore I know exactly which band is my protein of interest” so you thought…


The protein size should not be the only parameter used to validate a protein. The migration of proteins through a gel is affected by other factors than their size.

The mode of molecular separation also depends on the conditions under which an electrophoretic run takes place. The separation of proteins depends upon the gel pore size of the support matrix. Polyacrylamide has a small pore size and is ideal for separating most proteins and smaller nucleic acids. 

As a result, the molecular weight and the protein migration in WB will not always match: the same protein might migrate differentially on gels of different constitutions.  

Suitable examples are the calcium-binding proteins calsequestrin-1 and 2 (CSQ1 and CSQ2). CSQ1 and CSQ2 have predicted molecular weights of 47.8 and 45.3 kDa, respectively. However, in Tris-HCl SDS/PAGE gels run at pH 8.3, they migrate at ∼63 and 55 kDa, respectively (Murphy et al. 2009a). When using Bis-Tris-Cl gels, run at pH 7.3, CSQ1 migrates faster (smaller molecular weight) than CSQ2. This is because of the denaturing conditions of the SDS that bind all the proteins giving them an overall negative charge, thus overriding the native charge that the proteins possess.

In conclusion, the molecular weight and migration in SDS/PAGE WB gels do not always match; hence, the apparent size should not be the only parameter used to recognize a protein.


Read more about WB and antibody applications



  • Bass JJ et al., (2017) Review: an overview of technical considerations for Western blotting applications to physiological research. Scand J Med Sci Sports 27: 4–25

  • Murphy RM. et al., (2011a) Quantification of calsequestrin 2 (CSQ2) in sheep cardiac muscle and Ca2+-binding protein changes in CSQ2 knockout mice. Am J Physiol Heart Circ Physiol. 300:H595–H604. Nature Communications 9: 4130

  • Robyn MM. et al., (2013) Important considerations for protein analyses using antibody-based techniques: down-sizing Western blotting up-sizes outcomes. J Physiol. 591(Pt 23): 5823–31.

  • Yang, CR. et al., (2015) Deep proteomic profiling of vasopressin-sensitive collecting duct cells. I. Virtual Western blots and molecular weight distributions. Am. J. Physiol. Cell Physiol. 309, C785–C798.