The nascent form of PARP1 (116 kDa) can be cleaved by caspase-3 to produce its mature form (89 kDa) (Joshi et al., 2014 Nicholson et al., 1995). When these isoforms co-exist within a cell, an antibody may detect multiple bands on a Western blot.įor an example, let’s talk about poly(ADP-Ribose) polymerase 1 (PARP,1 also referred to as PARP) which is a 116 kDa nuclear enzyme involved in DNA damage signaling, chromatin remodeling and epigenetic regulation of gene expression (Ciccarone et al., 2017). Lastly, multiple protein isoforms can be derived from a single gene through alternative splicing, a mechanism cells employ to generate a multitude of protein diversity from a limited array of genes (Baralle and Giudice, 2017 Ule and Blencowe, 2019). In cases like these, it is common to detect multiple bands. For example, caspase-1 is activated through self-cleavage and will then cleave pro-IL-1β and pro-IL-18 giving rise to mature interleukin proteins (Malik and Kanneganti, 2017). These PTMs are common and indispensable cellular events that are critical for protein maturation and function (Eifler and Vertegaal, 2015 Liu et al., 2016). Another possibility for multiple bands is a result of post-translational processing such as cleavage or modifications (PTM), including but not limited to, phosphorylation, acetylation, glycosylation, ubiquitination, SUMOylation, succinylation, and methylation. As such, antibodies developed against intact and fragmented portions of a protein will result in multiple bands that often appear as band smearing. In other words, protein homeostasis (proteostasis) maintains a viable protein pool by constantly undergoing synthesis and degradation (Rahman and Sadygov, 2017). All proteins will undergo biogenesis, folding, trafficking, and degradation, however, the rate of turnover varies from protein-to-protein depending on its half-life and cellular processes such as its function and subcellular location (Rahman and Sadygov, 2017). Alternatively, multiple bands may result from the detection of a protein during turnover, as protein homeostasis is a dynamic process. For example, cardiac α- and β- myosin heavy chain isoforms share 93% sequence identity (Krenz et al., 2003), and the development of an antibody against the shared sequence of the family members will most likely produce multiple bands on a Western blot. One of the frequent challenges is that many proteins, particularly those proteins belong to the same families, can contain the same isotope and result in the development of multiple bands. There are numerous reasons for non-specific binding of an antibody to multiple proteins. It is possible that multiple bands on a Western blot indicate non-specificity, as indicated in Figure 1D in my previous blog post (Blog#1). These extra bands bewildered me because it was unclear whether or not these antibodies were specific. In my research career of 30+ years, I have encountered hundreds of antibodies that have exhibited multiple banding properties, particularly with tissue lysates.
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