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Many crop species across the plant family contain high polysaccharide and polyphenols. The hydrocolloid mucilage is a complex mucopolysaccharide typically showing high viscosity when released in solution. Homogenate of high mucopolysaccharide plant samples usually imparts a slimy consistency that is most unwanted contaminants that interfere with cell fractionation and organelle purification, such as chloroplast and nuclear isolation. This kit provides a simple, rapid, and efficient method for isolating chloroplasts from viscous plant homogenate. The protocol takes about 30 min.

Researchers studying protein localization and trafficking now have a convenient tool: the MinuteTM Water-soluble and Water-Insoluble Protein Fractionation Kit for cells/tissues. This unique product allows for the rapid and efficient separation of these two key fractions from cells/tissues, microorganisms and plant tissues, providing enriched and easily detectable protein samples for downstream applications like western blotting, ELISA and immunoprecipitation. Water soluble fraction contains mainly cytosolic proteins and water-insoluble fraction contains mainly insoluble membranous structures such as plasma membrane, nuclear membrane and organelles. The kit offers researchers a novel and simple approach for understanding protein function and movement within the cellular landscape. If a protein target is known to be localized in water-soluble or water-insoluble fraction, the kit will facilitate detection, isolation and purification of the target protein by enrich corresponding fractions.

There are substantial evidences that support the existence of detergent-resistant membrane (DRM) subdomains in both animal and plant membrane systems, which is also referred to as lipid rafts. Lipid rafts are shown to have increased sphingolipid to protein ratio and higher cholesterol concentration. The lipid rafts are believed to play an important role in signal transduction and protein trafficking in eukaryotic cells. Traditional method for lipid raft isolation involves cold non-ionic detergent extraction followed by sucrose gradient ultracentrifugation. The protocol requires large amount of starting material (hundred grams amount) and specialized equipment in addition to lengthy protocol time. We have developed a rapid method for isolation of lipid rafts from plant tissues using only milligram amount of starting material and the protocol can be completed in about 1h without using ultracentrifugation.

The proteasome is a protein degradation system that requires metabolic energy and polymerization of ubiquitin that is different from lysosome-based protein degradation. The 26S proteasome is made up of two subcomplexes (a 20S proteasome and one or two19S regulatory particles). The proteasomes have been found mainly localized in the cytosol but also detected in the nucleus. Traditionally, ultracentrifugation and affinity isolation are the most common methods for the isolation of proteasomes. These methods, though relatively effective, are usually time-consuming with low yield. Many affinity-based methods require harsh elution conditions that may affect the activity of isolated proteasomes and limit certain downstream applications. In most cases, the methods can’t distinguish proteasomes derived from cytosol or nucleus. To overcome these shortcomings, we have developed spin-column-based kits for the enrichment of proteasome from nuclei (Cytosolic Proteasome Enrichment Kit is also available under Cat # PT-040). The protocol is simple and rapid with a high yield. The gentle protocol maintains the association of proteasomes with ubiquitin and other proteins and is useful for the studies of proteasome structure and function. The highly enriched proteasomes can also be used as a starting material for affinity purifications.

Diagnoses using exosomes derived from saliva have been attracting great attention in recent years due to the ease of sample collection. However, saliva samples are challenging when it comes down to exosome isolation. In addition to cells and cell debris, large amount of amylase, mucin and glycoprotein are present in saliva, and make the samples viscous and hard to manipulate. Pre-treatments, such as sonication and dilution, are often required. In many cases, the saliva samples can still be difficult to handle even after pre-treatments. This product is specifically designed to address these issues using the proprietary saliva filters. Highly viscous saliva samples can be converted to non-viscous solutions instantly by passing the samples through the filter. Exosomes can then be readily precipitated from as little as 100 µl saliva using the highly effective non-PEG-based reagent.

The plant Golgi apparatus plays an important role in the biosynthesis of cellular structural components and protein trafficking. However proteomic characterization of the Golgi has been hindered by limited methodologies for obtaining enriched/purified Golgi fraction. The traditional method for isolating Golgi apparatus depends upon the use of density centrifugation, which is tedious and time-consuming. A large amount of starting material is usually required. Plant Golgi stacks are notoriously labile and loss of stack integrity can occur if harsh homogenization is used. This means that assessment of purity by morphological means such as electron microscopy is difficult. This kit is designed to overcome certain disadvantages of traditional methods by employing spin column-based homogenization, differential centrifugation, and selective precipitation for the enrichment of the Golgi. The protocol is simple and straightforward and requires only a milligram amount of starting material. Significant enrichment of the Golgi fraction (2-3 folds) can be obtained.

An understanding of endomembrane processes and their biological implication is important for understanding plant growth and development. Endosomes are a heterogeneous collection of organelles in the sorting and delivery of internalized material from cell surface and the transport of materials from Golgi to lysosome or vacuole. In plant cells, two clearly defined endosomal compartments have been identified: the trans-Golgi network (early endosome equivalent) and the multivesicular body (late endosome equivalent). Traditionally, methods for isolation and purification of these two compartments are tedious and time-consuming involving sucrose gradient centrifugation. In some cases, sucrose gradient isolation is coupled with immunoaffinity purification and larger amount of starting materials are usually required (10-50 g). We have developed this kit using a spin-column-based format coupling with selective precipitation for enrichment of endosomal compartment. This approach is easy and rapid and only small amount of starting material is required. 

Red blood cells (RBCs) are essential components of the circulatory system, responsible for transporting oxygen and carbon dioxide throughout the body. Understanding their surface composition, particularly the diverse array of proteins is crucial for research in blood biology and disease. This protocol offers a rapid and efficient method for isolating native plasma membranes (PM) vesicles from RBCs with associated membrane proteins. The method utilizes differential centrifugation and a specialized protein extraction powder for gentle RBC lysis while enriching for PM fractions. Importantly, this approach preserves the native protein conformation and avoids the use of detergents, facilitating for downstream studies of membrane-associated proteins. The entire protocol can be completed in approximately 1 hour, yielding highly enriched PM with minimal contamination from intracellular components like spectrin and hemoglobin.

A chloroplast, delimited by a two-membrane envelope, is a type of plastid that serves as the site of photosynthesis. The photosynthetic function is based on developing an operational extensive internal membrane network, called the thylakoids, and on enzymatic processes in the chloroplast matrix, called the stroma. Thylakoid membrane is structurally different from the chloroplast envelope, and their biogenesis depends on biosynthetic and transporting activities specific to the chloroplast envelope. This kit is designed to fractionate chloroplasts into three parts (thylakoid membrane, envelope membrane, and stroma) using a specialized filter cartridge and differential centrifugation (without ultracentrifugation). Isolated thylakoids are in native forms and can be used for functional studies and the studies of subcellular localization of proteins using Western blotting/Mass spectrometry. The buffers in the kit are detergent and EDTA-free. The protocol can be completed in about one hour.