Cell disruption protocols

The composition of the lysis solution is dictated by the nature of the proteins under study and the subsequent techniques applied to the sample. One of the major choices to be made is whether or not a detergent is required at this stage. If the membrane and soluble fractions are to be separated the initial cell disruption protocol should not include a detergent, as many of the membrane proteins would be solubilized. In this case physical disruption of the cells should be used (e.g., sonication of cells or homogenization of tissues). The choice of lysis conditions is a vital consideration in this work, as proteins need to be solubilized while preservation of posttranslational modifications, inhibition of proteases, maintenance of protein-protein interactions, and, if an immunoaffinity purification step is to be performed, suitability for the antibody to function are essential. For example, SDS is very good at solubilizing membrane proteins but... 

Machen A, Kobayashi M, Connelly MR, Wang YF (Wayne). Comparison of heat inactivation and cell disruption protocols for identification of mycobacteria from solid culture media by use of vitek matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 2013 51(12) 4226-9. 

Protocol 2.8 Lysis using cell disruption or sonication... 

In order to check the effectiveness of the cryoprotectants, neurons were cryopreserved with regular culture medium (without cryoprotectants). Recovering cells were not observed in microscopic observations when cryopreservation was carried out using the same experimental protocol (Figure 4). In case of cryopreservation in the absence of cryoprotectants, the ice around the cells disrupts the cell membrane. 

The first step is not required for chemically synthesized products, otherwise prior cell disruption and organelle separation are required to yield cell-free extract from which the desired biomacromolecule can be purified from its natural enviromnent. Total cellular or tissue proteins may be solubilized and assayed prior to purification (Shaw, 1998). Different approaches are available to lyse cells (http //expasy.cbr.nrc.ca/ch2d/protocols/). An approach can be as gentle as adding a surfactant or subjection to an osmotic shock, or can be more energetic such as ultra-sonification, bead beater or French press. Table 3.1 lists some of common methods for cell disruption. 

Buffers containing SDS for cell lysis (Protocols lA and IB) are incompatible with some subsequent methods of analysis, for example non-denaturing PAGE (Protocol 12) or immunopredpitation under non-disruptive conditions (Protocol 14B). In order to study physiological interactions, it is necessary to perform cell lysis in buffers containing no detergents or mild non-ionic detergents, such as Triton X-100 or Nonidet P-40. 

Amperometry at single PC 12 cells has also been used in conjunction with a genetic cell transfection protocol to examine the effects of toxin expression on basal and evoked exocytosis. PC 12 cells have been transfected with the specific endoprotease Botulinum neurotoxin Cl light chain (BoNT/Cl), which cleaves the proteins syntaxin and SNAP-25 [5], The molecular dissection of the mechanisms underlying exocytosis has been motivated by the SNARE hypothesis, which postulates that exocytosis requires the assembly of the plasma membrane proteins syntaxin 1, SNAP-25, and the vesicle associated membrane protein (VAMP) into a complex [5], This SNARE complex then acts as a receptor for cytosolic components of the proposed fusion machinery. Direct evidence for the role of the SNARE proteins in neurotransmission comes from molecular genetic studies in which syntaxin and VAMP have been shown to be required for neurotransmission in Drosophila [47 9] and Caenorhabditis elegans [50,51]. To assess the effects of the disruption of SNARE proteins on exocytosis in PC 12 cells, amperometry has been used in conjunction with a genetic cell transfection assay to establish a... 

Slomiany B.L. and Slomiany A. (2002) Disruption in gastric mucin synthesis by Helicobacter pylori lipopolysaccharide involves ERK and P38 mitogen-activated protein kinase participation. Biochem Biophys Res Commun 294, 220-224 Snapp E., Altan N. and Lippincott-Schwartz J. (2003) Fluorescence Recovery After Photo-bleaching. In Current Protocols in Cell Biology. (Bonifacino, J., Dasso, M., Harford, J., Lippincott-Schwartz, J., Yamada K., Morgan K.S., eds.) Unit 21.1 John Wiley Sons, Inc., New York... 

Wittrup and Belting have in several studies on CPP-mediated DNA delivery established protocols for fluorescence assisted cell sorting (FACS) analysis to obtain reliable, quantitative data on CPP-DNA complex uptake in cultured cancer cells (9). Also, procedures for co-localization studies with known markers of various endocytotic pathways using confocal microscopy are described as well as the expression of dominant negative dynamin (GTPase deficient dynamin-2) to evaluate the dynamin dependence of the uptake mechanism. The use of various drugs commonly used to disrupt endocytosis is discussed, especially with regard to their limited specificity (9). 

Prior to total RNA extraction, sample lysis procedures have to be performed. Lysis conditions are very important for the success of the RNA extraction and depend strongly upon the sample used. Due to great diversity, the biological sample can be pulverized, homogenized, sonicated, or otherwise disrupted to yield a mixture that contains cells, subcellular components, and other biological debris in an aqueous buffer or suspension. Here is described the protocol for the Trizol method of RNA extraction. 

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