Cell Lysis Solutions
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 differing 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.
This 50-page handbook provides protocols and technical and product information to help maximize results for protein/gene expression studies. The handbook provides background, helpful hints and troubleshooting advice for cell lysis, protein purification, cell fractionation, protease inhibitors and protein refolding. The handbook is an essential resource for any laboratory studying protein/gene expression.
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 (radio-immunoprecipitation 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: 25mM Tris•HCl, pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% sodium dodecyl sulfate (SDS).
When the immediate downstream application for a lysate is a protein affinity purification experiment such as immunoprecipitation (IP) or co-immunoprecipiation (Co-IP), 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 M-PER 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. Pierce IP Lysis Buffer was formulated to provide good results for IP overall.
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.
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 25mM Bicine, 150mM 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.
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(-) 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.
Y-PER Yeast Protein Extraction Reagents penetrate 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 Reagents provide higher yields and greater flexibility for use in current proteomics workflows.
Two formulations have been developed. Y-PER Reagent is high salt (>300mM) 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. Y-PER Plus Dialyzable Reagent, (Part No. 78999) is a phosphate-free, low-salt formulation with a dialyzable detergent. It is validated for use primarily with S. cerevisiae.
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).
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.
For Research Use Only. Not for use in diagnostic procedures.