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Cell lysis using detergents
Detergent cell lysis is a milder and easier alternative to physical disruption of cell membranes, although it is often used in conjunction with homogenization and mechanical grinding. Detergents break the lipid barrier surrounding cells by solubilizing proteins and disrupting lipid–lipid, protein–protein and protein–lipid interactions. Detergents, like lipids, self-associate and bind to hydrophobic surfaces. They are comprised of a polar hydrophilic head group and a nonpolar hydrophobic tail, and are categorized by the nature of the head group as either ionic (cationic or anionic), nonionic, or zwitterionic. Their behavior depends on the properties of the head group and tail.
Unfortunately, there is no standard protocol available for selecting a detergent to use for membrane lysis. In general, nonionic and zwitterionic detergents are milder and less denaturing than ionic detergents and are used to solubilize membrane proteins where it is critical to maintain protein function and/or retain native protein–protein interactions for enzyme assays or immunoassays. CHAPS, a zwitterionic detergent, and the Triton-X series of nonionic detergents are commonly used for these purposes. In contrast, ionic detergents are strong solubilizing agents and tend to denature proteins, thereby destroying protein activity and function.
The choice of detergent for cell lysis also depends on sample type. Animal cells, bacteria and yeast all have different requirements for optimal lysis due to the presence or absence of a cell wall. Because of the dense and complex nature of animal tissues, they require both detergent and mechanical lysis. In addition to the choice of detergent, other important considerations for optimal cell lysis include the buffer, pH, salt concentration and temperature. Consideration should be given to the compatibility of the chosen detergent with downstream applications. If the detergent used for lysis must be removed, then a dialyzable detergent should be selected.
RIPA buffer is a popular choice for lysis and protein extraction of mammalian cells or soft tissue. Originally named after the assay method for which it was developed (radioimmunoprecipitation assay), RIPA buffer is effective when the immediate downstream application is SDS-PAGE (denaturing polyacrylamide gel electrophoresis). The formulation includes two ionic detergents and one nonionic detergent in Tris buffer: 25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP40, 1% sodium deoxycholate and 0.1% sodium dodecyl sulfate (SDS).
Protein extraction using RIPA buffer. RIPA buffer enables rapid and effective extraction of cytoplasmic, membrane and nuclear proteins from cultured mammalian cells (plated cells, pelleted suspension cells) and tissues. As a western blotting standard, it is compatible with protease and phosphatase inhibitors, minimizes non-specific protein binding, and is compatible with many downstream applications, including reporter assays, protein assays and immunoassays.
When the immediate downstream application for a lysate is a protein affinity purification experiment such as immunoprecipitation (IP) or co-immunoprecipiation (CoIP), it is especially important to ensure that the lysis reagent does not contain denaturants or components that might interfere with the antibody binding or protein interactions of interest. RIPA buffer, despite its name, is not always the best choice for immunoprecipitation experiments because it contains SDS. Our MPER buffer (see next) is one alternative. Another option is to formulate your own lysis buffer or to test alternative commercial solutions to find the one that provides the best balance between extraction yield and IP success. Thermo Scientific Pierce IP Lysis Buffer was formulated to provide good results for IP overall.
Watch these videos to learn more about immunoprecipitation
Thermo Scientific M-PER Mammalian Protein Extraction Reagent was developed as an effective yet milder alternative to RIPA buffer. M-PER reagent uses a non-denaturing detergent to prepare total cell lysate that is compatible with many downstream assays, including immunoassays, enzyme assays and a variety of common reporter assays. Lysis can be performed directly in culture plates and is completed in only 5 minutes. Furthermore, significantly more protein can be obtained with this method compared with freeze/thaw and sonication.
Comparison of M-PER reagent with freeze/thaw cycles, sonication and Brand P lysis buffer. COS-7 cells grown in 100-mm plates at full confluency were washed once with 10 mL of PBS, scraped with 1 mL of PBS, and centrifuged at 5000 rpm for 5 minutes to collect the cells. The cell pellets were resuspended in 0.5 mL of respective extraction reagents and subjected to total protein extraction. For freeze/thaw cycles, the cell suspension in PBS was frozen in a dry ice and isopropanol bath for 10 minutes and thawed in a 37°C water bath. The freeze/thaw cycle was repeated three times. For sonication, the cell suspension was sonicated for 2 minutes with a 50% pulse using a Branson Sonifier 450 Sonicator. For extraction with M-PER reagent and Brand P lysis buffer, the cell suspensions were shaken for 5 minutes. The cell debris was removed by centrifugation at 13,000 rpm for 5 minutes and the supernatants were assayed for protein concentration by the BCA method.
Thermo Scientific T-PER Tissue Protein Extraction Reagent is designed for total protein extraction from mammalian tissue samples, including heart, liver, kidney and brain. The T-PER reagent utilizes a non-denaturing detergent in 25 mM bicine, 150 mM NaCl (pH 7.6), and is used in conjunction with mechanical or manual homogenization. The resulting cell lysate, like the lysate prepared with M-PER reagent, is compatible with many functional assays.
Protein extraction from various tissue types. Duplicate tissue samples were weighed, resuspended in 1:10 to 1:20 w/v T-PER reagent and disrupted in a chilled Dounce or benchtop tissue homogenizer. The resulting lysates were centrifuged at 10,000 x g for 5 minutes and the supernatant was collected. The protein concentration of each lysate was determined using the Thermo Scientific Pierce BCA Protein Assay (Part No. 23227) to determine protein yield per milligram of starting tissue.
Thermo Scientific B-PER Bacterial Protein Extraction Reagents gently lyse E. coli and other species of bacterial cells and effectively extract soluble native and recombinant proteins. B-PER reagents have been used for Gram-negative bacteria, S. aureus, H. pylori and E. coli strains BL21(D3), JM109, DH5a and M15. The reagent is also suitable for certain Archaebacteria species and cultured insect cells. Extraction does not require expensive equipment and can be performed in less than 10 minutes. B-PER reagent removes soluble protein from inclusion bodies and can be used to purify intact inclusion bodies whose less soluble proteins can be extracted by other means.
Several different ready-to-use formulations of B-PER reagent are available for different lysis needs. These include formulations in Tris buffer or PBS, and those with and without lysozyme and DNase I enzymes. The B-PER direct formulation is optimized for direct (homogenous) lysis of cells in 96-well culture plates, facilitating high-throughput screening assays.
B-PER Complete Bacterial Protein Extraction Reagent provides better compatibility with purification of 6xHis and GST fusion proteins. E. coli ER2566/pLATE51-Klenow, ER2566/pGST-Syk and ER2566/pGST-CC-StpA cell pellets (0.5 g) were resuspended in 2.5-mL aliquots of B-PER Complete reagent (B-PER) or EMD Millipore™ BugBuster™ Master Mix (Bug) with gentle vortexing for 15 minutes at room temperature. Insoluble cell debris was removed by centrifugation at 16,000 x g for 20 min at 4°C. 6xHis-Klenow protein was purified using Thermo Scientific HisPur Ni-NTA Superflow Agarose. GST-Syk and GST-StpA proteins were purified using Thermo Scientific Pierce Glutathione Agarose. C = control (E. coli lysate from before induction with IPTG); S = soluble protein (lysate after induction with 0.1 mM IPTG); E = elution fraction after protein purification.
Y-PER reagent disrupts yeast cell wall and plasma membrane. Cells of Saccharomyces cerevisiae strain DY150 after lysis with Y-PER Yeast Protein Extraction Reagent. Various points of perforation are visible, and these allow for extraction of cytosolic proteins.
Yeast cell lysis solutions
Thermo Scientific Y-PER Yeast Protein Extraction Reagent penetrates the tough yeast cell wall, perforating the cell wall and membrane and extracting soluble protein without completely damaging overall cell structure. Traditionally, yeast protein extraction required mechanical disruption with glass beads, making small-scale extraction difficult. Y-PER reagent provides higher yields and greater flexibility for use in current proteomics workflows.
Two formulations have been developed. Y-PER reagent is high salt (>300 mM) and is effective for S. cerevisiae, S. pombe, C. albicans and P. pastoris (as well as various Gram-positive and Gram-negative bacteria). Although the detergent is compatible with various downstream methods, it is not dialyzable so cannot be removed in those cases where incompatibility exists. Thermo Scientific Y-PER Plus Dialyzable Yeast Protein Extraction Reagent (Part No. 78999) is a phosphate-free, low-salt formulation with a dialyzable detergent. It is validated for use primarily with S. cerevisiae.
Insect cell lysis solutions
Thermo Scientific I-PER Insect Cell Protein Extraction Reagent enables gentle extraction of soluble protein from baculovirus-infected insect cells grown in suspension of monolayer culture (both Sf9 and Sf21 cells). The reagent maintains functionality of extracted proteins and is directly compatible with downstream applications such as protein assays, western blotting and His-tagged protein purification (IMAC).
Affinity purification of 6xHis Cyclin B1 from insect cell extract. Baculovirus-infected Sf9 cells were harvested and lysed with I-PER reagent. The extract was directly loaded onto a nickel-chelated agarose (IMAC) column and purified. Protein samples were separated by SDS-PAGE, and the gel was stained with Thermo Scientific GelCode Blue Stain Reagent.
Plant tissue cell lysis solutions
Thermo Scientific P-PER Plant Protein Extraction Reagent Kit offers an exclusive innovation for performing plan cell lysis and subsequent protein extraction. Plant cells are notoriously difficult to lyse and extract for proteomics work because their tough cell walls and substantial polysaccharide content. The kit includes an organic lysing reagent and two aqueous reagent which, in conjunction with mild mechanical agitation, effective extract plant protein. The method has been validated for use with multiple plan organs (leaf, stem, root, seed and flowers); multiple plant species (Arabidopsis, tobacco, maize, soybeans, peas, rice and spinach); and fresh, frozen and dehydrated tissue sources.
Protocol summary for the P-PER Plant Protein Extraction Reagent kit.
- Walker JM (2009) The Protein Protocols Handbook. Third Edition. New York (NY): Springer-Verlag New York, LLC.
For Research Use Only. Not for use in diagnostic procedures.