Fancy reagents and the wisdom of decades are facilitating the isolation and collection of subcellular parts and proteins. With improvements of the technology driven by proteomic studies, obtaining proteins exclusively from specific parts of the cell is now a standard procedure. Refinements and further sophistication are still to come, but the kits and protocols of today are leaps and bounds ahead of the limited capabilities of yesteryear.
The need to pull apart the cell’s organelles and proteins was considered so important that the 1974 Nobel Prize in Medicine was awarded to Claude de Duve, one of three researchers who played a primary role in promoting the study of “the structural and functional organization of the cell,” according to the awarding committee. While studying methods of cell fractionation with the centrifuge, de Duve discovered the lysosome and peroxisome. Since then, many researchers have built upon the foundation of de Duve’s work.
Advancements in fractionation techniques are more important these days, since researchers are usually most interested in proteins available in lower quantities. With these proteins ensconced in certain cellular structures, companies have developed reagents that can solubilize the molecules that make up those structures. Packaged into kits, extracting your proteins of interest is now convenient and reliable.
Many kits target the cytosol, plasma membrane, nucleus, cytoskeleton, and organelles, yielding proteins from these structures through a series of steps. Using reagents based upon the different solubilities of these structures and centrifugation, the kits draw out the proteins you’re after. You can even purchase kits that target different proteins located in the same structure. For example, while one kit extracts receptor proteins in the membrane, other kits may specialize in other types of membrane proteins. If you want to use the proteins for subsequent studies, look for a kit that comes with a protease to prevent denaturing of the proteins.
While cellular fractionation used to mean long hours of centrifugation, affinity enrichment, and homogenization, today’s kits can finish the isolation job in just a couple of hours. Check out the items below. You’re sure to find a product that will streamline your proteomic studies.
The ProteoExtract Subcellular Proteome Extraction Kit is a tool for the differential extraction of proteins from adherent or suspension tissue culture cells according to their subcellular localization. The sequential extraction steps yield fractions containing cytosolic proteins, plasma membrane and organelle proteins, soluble nuclear proteins, and finally cytoskeletal and nuclear matrix proteins. To preserve the protein profile and increase spot resolution in 2-D gel electrophoresis, the kit also contains Protease Inhibitor Cocktail and the proprietary non-specific Benzonase® Nuclease.
One of the challenges of functional proteomics is separation of complex protein mixtures for quantitative and differential subcellular localization analysis. This necessitates standardized and repeatable operation procedures to isolate subcellular proteomes from tissues and cells. The Chemicon Compartmental Protein Extraction Kit addresses the challenge by providing an innovative, easy-to-perform, and cost-effective method to sequentially isolate cytoplasmic, nuclear, membrane, and cytoskeleton proteins from mammalian tissues and cells based on a proprietary technique. This kit contains enough reagents to enrich four compartmental proteins from 5 grams tissues or approximately 125 million cells.
This kit is based on the separation of membrane proteins by temperature-dependent phase partitioning using the detergent Triton X-114 (Bordier 1981, Santoni et al. 2000). After a final centrifugation step, the sample produces an upper aqueous phase and a lower detergent-rich phase. An insoluble pellet is also formed and is a source of more complex membrane proteins. Proteins extracted into the two phases are treated using the ReadyPrep 2-D cleanup kit (also provided in this kit) to remove agents in the extraction process that interfere with IEF.
