Sample prep is common (and often overlooked) source of error when it comes to troubleshooting Western blots. One step in particular — fractionation — is suggested to reduce the “cellular debris,” but may in reality be harming your sample.
“Cellular debris” is a misnomer applied to cell components, such as the nuclei and membranes, that commonly form a pellet during centrifugation. These structures are often discarded when the protein of interest is thought to exist primarily the subcellular fraction, which is enriched in the supernatant.
However, this assumption leads many researchers to arbitrarily fractionate all of their Western blot samples, perhaps using a standard lab protocol for months or years. Many scientists think the “cellular debris” hinders the quality of the blot, and does not make up an important portion of their sample.
In one 2014 article in Expert Rev Proteomics, Rajeshwary Ghosh et al. note that sample quality may suffer from misguided notions about cellular debris. “A misconception is that the ‘cellular debris’ of a homogenate interferes with the detection and quantification of the target protein,” the authors write.
They go on to note that cellular debris contains high levels of many important proteins, such as myosin and calsequestrin-2 in skeletal muscle, which may be improperly excluded from analysis because of ill-advised protocols.
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In a report in J Physiol (2013), authors Robyn Murphy and Graham Lamb conclude that “correct results are most easily obtained when no tissue fractionation is undertaken. The assumed need to undertake fractionation is often unwarranted because even lowly abundant proteins can be successfully detected by simply using less sample and more sensitive signal detection.”
Besides reducing the amount of your sample, carefully considering your lysis buffers can eliminate any further need for fractionation. For instance, Ghosh et al. suggests the addition of a DNase before lysis to prevent “steaks” that sometimes appear in SDS-PAGE gels when running cell culture sample.
If you still think fractionation is necessary for the success of your experiment, be sure to save all pellets formed during the centrifugation steps. Run a separate blot with these lysates alongside some of your cytoplasmic samples, and probe for your target. You may be surprised to find you've been discarding a large portion of your protein of interest unknowingly until now.
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Ghosh, R., Gilda, J. E., & Gomes, A. V. (n.d.). The necessity of and strategies for improving confidence in the accuracy of western blots. Expert Rev Proteomics, 11(5), 549-560. https://doi.org/10.1586/14789450.2014.939635
Murphy, R. M., & Lamb, G. D. (2013). Important considerations for protein analyses using antibody based techniques: down-sizing Western blotting up-sizes outcomes. The Journal of Physiology, 591(23), 5823–5831. https://doi.org/10.1113/jphysiol.2013.263251