Mon. Dec 9th, 2019

QIAGEN: Qproteome Cell Compartment Handbook

2 min read
Two-Step Affinity Purification System Handbook

Two-Step Affinity Purification System Handbook

Introduction

Eukaryotic cells are complex, well-ordered, and highly structured systems. The Cell Compartment Kit is designed for fast and easy subcellular fractionation of intact eukaryotic cells. By sequential addition of different extraction buffers to a cell pellet, proteins in the different cellular compartments can be selectively isolated (see Table 1, Figure 1). Principle and procedure Extraction Buffer CE1 is added to cells and selectively disrupts the plasma membrane without solubilizing it, resulting in the isolation of cytosolic proteins. Plasma membranes and compartmentalized organelles, such as nuclei, mitochondria, and the endoplasmic reticulum (ER), remain intact and are pelleted by centrifugation. The pellet from the first step is resuspended in Extraction Buffer CE2, which solubilizes the plasma membrane as well as all organelle membranes except the nuclear membrane. After solubilization, the sample is centrifuged. The supernatant contains membrane proteins and proteins from the lumen of organelles (e.g., the ER and mitochondria). The pellet consists of nuclei. In the next step nuclei are solubilized using Extraction Buffer CE3 in which all soluble and most membrane-bound nuclear proteins are extracted. Addition of Benzonase® allows the release of proteins tightly bound to nucleic acids (e.g., histones). After another centrifugation, Extraction Buffer CE4 is used to solubilizes all residual — mainly cytoskeletal — proteins in the pellet. Fractions 1 to 3 contain proteins in their native state. Extraction Buffer CE4 is strongly denaturing and not compatible with isoelectric focusing. Proteins in all fractions must be desalted (e.g., by acetone precipitation, see page 12) before further analysis using isolectric focusing. Starting material for one fractionation procedure is 5 x 106 cells. The procedure has been used successfully for several different mammalian cell lines including HeLa, Jurkat, NIH-3T3, HEK293, and Cos. Table 2 gives an overview of expected protein yields using different cell lines. Subcellular fractionation of proteins enables: „Enrichment of low-abundance species „Definition of the subcellular localization of enzymes, regulatory, and structural proteins „Monitoring of compartmental redistribution of biomolecules under basal and stimulated conditions

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