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Organelle isolation Single cell analysis Tissue analysisEndoplasmic Reticulum Isolation: An Optimized Approach into Cells and Mouse Liver Fractionation
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
Detection of Cytoplasmic and Nuclear Circular RNA via RT-qPCR
Circular RNA (circRNA) is an intriguing class of non-coding RNA that exists as a continuous closed loop. With the improvements in high throughput sequencing, biochemical analysis, and bioinformatic algorithms, studies on circRNA expression became abundant in recent years. However, functional studies of circRNA are still limited. Subcellular localization of circRNA may provide some clues in elucidating its biological functions by performing subcellular fractionation assay. Notably, circRNAs that are predominantly found in the cytoplasm are more likely to be involved in post-transcriptional gene regulation, e.g., acting as micoRNA sponge, whereas nuclear-retained circRNAs are predicted to play a role in transcriptional regulation. Subcellular fractionation could help researchers to narrow down and prioritize downstream experiments. The majority of the currently available protocols describe the steps for subcellular fractionation followed by western blot analysis for protein molecules. Here, we present a protocol for the subcellular fractionation of cells to detect circRNA via RT-qPCR with divergent primers. Moreover, detailed steps for the generation of specific circRNAs-enriched cDNA included in this protocol will enhance the amplification and detection of low-abundance circRNAs. This will be useful for researchers studying low-abundance circRNAs.
Key features
• This protocol builds upon the method developed by Gagnon et al. (2014) and extends its application to circRNA study.
• Protocol for amplification of low levels of circRNA expression.
• Analysis takes into consideration the ratio of cytoplasmic RNA concentration to nuclear RNA concentration.
Graphical overview
Membrane Flotation Assay
Isolation of Tomato Fruit Chromoplasts and Determination of ATP Levels
Small-scale Subcellular Fractionation with Sucrose Step Gradient
Subcellular fractionation is a very useful technique but usually a large number of cells are required. Because we needed subcellular fractionation of transiently-transfected cells, we developed a protocol for smaller numbers of cells. Our protocol for the subcellular fractionation is based on the protocol published by de Araújo and Huber (de Araujo et al., 2007), although substantial modifications have been made according to our experiences and information from personal communications. As optimal conditions seem to vary between cell lines, we advise to further modify the protocol to optimize for individual experiments. Our method is simple but sufficient for analysis of integral membrane proteins or proteins anchored to organelles by glycosylphosphatidylinositol or other lipid anchors, e.g. prion protein. However, proteins non-covalently attached to membranes or membrane proteins of organelles seem to be more prone to dissociation from the organelles during preparation and, if these proteins are the object of study, further modifications might be necessary.
Unlike in a continuous gradient, where a protein of interest is scattered over a wide range, step-gradient fractionation is advantageous in detection of relatively small amounts of proteins from small-scale experiments, because it concentrates the protein of interest in one fraction, if an appropriate combination of sucrose concentrations is used.
Cell Fractionation of Pseudomonas aeruginosa
Preparation of Pre- and Post-synaptic Density Fraction from Mouse Cortex
Isolation of Growth Cones from Mouse Brain
Subcellular Fractionation of Mouse Brain Homogenates
Subcellular Fractionation of Cultured Human Cell Lines
