Background

The endoplasmic reticulum (ER) is a continuous membrane system that forms a series of flattened sacs within the cytoplasm of eukaryotic cells and serves multiple functions. Some of its most relevant roles include the synthesis, folding, modification, and transport of proteins (Schwarz and Blower, 2016). The ER was first observed by electron microscopy in 1945 by Porter and Claude (Porter et al., 1945), but its isolation as a distinct organelle was not achieved until 1959 by Palade and Siekevitz (Siekevitz and Palade, 1959). In this study, authors used differential centrifugation to separate ER membranes from other cellular components. This method was later refined by adding density gradient centrifugations to obtain cleaner (non-other contaminating organelles marker) fractions (Lee et al., 2010).

The isolation of the ER has facilitated and enhanced the understanding of its structure, function, and interactions with other organelles. Some of these studies revealed that the ER forms contact sites with other membranes, such as plasma membrane–associated membranes (PAMs) (Suski et al., 2014) and mitochondria-associated membranes (MAMs) (Missiroli et al., 2018), which mediate transport of proteins, lipids, and metabolites (Ventura and Isabel Hernández-Alvarez, 2022).

However, despite being a highly common technique, the isolation of ER has some limitations. Firstly, it requires a large amount of starting material (usually several grams of tissue or cell number) and takes a long time to complete (6 h in cells because of the time needed to recover the starting material from the plates). Secondly, existing protocols may not be suitable for all tissues or cell types that have different membrane properties or distributions. Thirdly, it is quite difficult to obtain pure samples, and thus achieving highly enriched fractions can be challenging.

Most already existing methods are able to isolate ER indirectly, but only a very small number of protocols are dedicated to the extraction of this particular organelle (Croze and Morré, 1984). This scenario can be found in some of the published protocols that use mouse liver, such as the ones described by Wieckowski et al. (2009) or Suski et al. (2014), where they propose a method to isolate specific areas of the ER, such as MAMs and PAMs, respectively. This fact can also be a limiting factor.

In regard to HeLa cells, no protocols were found to specifically extract the ER as well. Moreover, some protocols that work with similar adherent cells require a large number of dishes (over 50 dishes) (Wieckowski et al., 2009; Williamson et al., 2015), which complicates the isolation and the previous cell culture work. Additionally, a similar situation happens with subcellular fractionations that use liver tissue. According to published protocols, fractionations of liver tissue are performed using rat liver rather than mouse liver, and they usually require from 8 to 10 g of tissue (Suski et al., 2014). When extrapolated to a mouse model, five or six animals for every subfractionation are needed.

Since our laboratory is mainly focused on the study of mitochondrial dynamics, ER–mitochondria contacts, and its relationship with metabolic diseases (Hernández-Alvarez et al., 2019), a protocol that overcame some of these limitations was necessary. Hence, in this article, we provide an optimized and upgraded version of already existing protocols for ER isolation achieving a high throughput using the minimum amount of starting sample. Furthermore, the protocols described below are optimized for time efficiency and designed for seamless execution in any laboratory, eliminating the need for special/patented reagents. Other protocols do not report the yield of ER.