Cell Biology


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0 Q&A 655 Views Sep 5, 2023

The subfractionation of the endoplasmic reticulum (ER) is a widely used technique in cell biology. However, current protocols present limitations such as low yield, the use of large number of dishes, and contamination with other organelles. Here, we describe an improved method for ER subfractionation that solves other reported methods' main limitations of being time consuming and requiring less starting material. Our protocol involves a combination of different centrifugations and special buffer incubations as well as a fine-tuned method for homogenization followed by western blotting to confirm the purity of the fractions. This protocol contains a method to extract clean ER samples from cells using only five (150 mm) dishes instead of over 50 plates needed in other protocols. In addition, in this article we not only propose a new cell fractionation approach but also an optimized method to isolate pure ER fractions from one mouse liver instead of three, which are commonly used in other protocols. The protocols described here are optimized for time efficiency and designed for seamless execution in any laboratory, eliminating the need for special/patented reagents.

Key features

• Subcellular fractionation from cells and mouse liver.

• Uses only five dishes (150 mm) or one mouse liver to extract highly enriched endoplasmic reticulum without mitochondrial-associated membrane contamination.

• These protocols require the use of ultracentrifuges, dounce homogenizers, and/or Teflon Potter Elvehjem.

• As a result, highly enriched/clean samples are obtained.

Graphical overview

0 Q&A 4626 Views Jun 5, 2019
Cytosolic rRNAs are highly dynamic and can be degraded under conditions such as apoptosis, starvation and magnesium depletion. The degradation is also related to their specific localization, as fractions of cytosolic ribosomes are localized on the surfaces of intracellular organelles, such as endoplasmic reticulum (ER) and mitochondria. Such localized translation facilitates translocation of nascent proteins into these organelles co-translationally, contributing to fast responses to cellular stresses and precise regulations of the organelle. Here, we describe a protocol to establish the in organello system to investigate rRNA degradation on mitochondrial outer membrane or ER. The protocol consists of organelle isolation, rRNA degradation on organelles and agarose gel electrophoresis to examine the remaining rRNAs.

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