Cell disruption sonic

In general, the physical structure of the tissue must be broken down mechanically followed by an extraction procedure, before the sample can be analyzed. Homogenization using blenders, probe homogenizers, cell disrupters, sonicators, or pestle grinders is particularly useful for muscle, liver, and kidney samples. Regardless of the method used for tissue disruption, the pulse, volume of extraction solvent added, and temperature should be validated and standardized in order to ensure reproducible analytical results. During cell disruption, care should be taken to avoid heat build-up in the sample, because the analyte may be heat labile. 

To check if PemB is surface exposed, E. chrysanthemi cells were subjected to proteolysis. Treatment of the cell suspension with trypsin, proteinase K or chimotrypsin at a concentration of 0.1 to 1 mg/ml for 1 h did not cause PemB proteolysis or its liberation into the medium. Cell pre-treatment with EDTA-lysozyme, which renders the periplasmic proteins accessible to proteases, gave no effect. PemB was also resistant to proteolytic digestion in extract of cells disrupted by sonication or in a French press. Only addition of Triton X-100 (up to 0.1%) causing formation of the micelles with PemB lead to a quick proteolyis of this protein (data not shown). In another approach to analyse the PemB exposition, bacterial cells were labelled with sulfo-NHS-biotin. This compound is unable to cross membranes and biotinylation... 

Protocol 2.8 Lysis using cell disruption or sonication... 

In order to cover glucuronidation reactions in incubations in microsomal fractions several modifications have been applied in order to optimize conditions. These comprise longer incubation times than necessary for oxidative reactions by cytochrome P450s, and use of modifiers, both to overcome the latency in activity due to the diffusional barriers of the endoplasmatic reticulum (Coughtrie and Fisher 2003 Csala et al. 2004). Modifiers used are detergents or the pore-forming peptide alamethicin (Fisher 2000). Also disruption of cells by sonication is applied (Ethell 1998). 

For the preparation of carbonyl-free solvents see Section 5.3.2.2.) The cells are washed three times in serum-free medium and harvested by scraping. The harvested cells are washed again with PBS, pH 7.4. Cell disruption is effected by light sonication and after centrifugation (600 x g, 15 min) the insoluble material is removed. The supernatant fraction is retained and the protein concentration assessed by the Lowry assay. 

Another use of cell disruption as a step in the analytical process is for obtaining a suspension of single cells — that can be used under optimal fermentation conditions — by ultrasonic disruption of cells manufactured in active dry wine yeast. Their potential was confirmed by comparing the elution profiles of non-sonicated and sonicated yeast sample dispersions obtained using two different field flow fractionation techniques [88]. 

A tubular sonicatlon device was recently reported by Borthwick et al. [93] (see Fig. 3.9). The device requires the addition of no chemical, enzyme or particles that might complicate the subsequent determination step. Furthermore, denaturatlon of target DMA or proteins for detection Is minimized as the device tolerates moderate temperature rises this allows the use of sensitive and specific Immunological detection methods on sonicated biological materials. Because the tubular device Is composed of a piezoelectric resonator made of several material layers, selection of an appropriate operating frequency Is essential to ensure proper performance (i.e. acceptable cell disruption efficiency). This device can be used for batchwise treatment of small sample volumes or In flow systems without the risk of hazardous aerosol formation inherent in probe sonloators. 

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... 

Sonication. Fermentor broths were reduced in volume via filtration to several liters or less. This reduced volume was then continuously circulated through a sonifer (Branson Cell Disrupter 185) ... 

Where the product must be captured from the intracellular environment, the first downstream processing steps are cell disruption and the removal of debris. Several methods have been used for disruption, including sonication, pressure homogenization, enzymatic treatment, and wet... 

Absorbance of sonicated cells Aliquot sonicated. S25 measured. Results comparable to dry weight. Sample same flask If flask Is large. Sonlcatlon takes time, extra manipulations, and an extra piece of equipment. Standard deviations high. Sonlcatlon may not disrupt cells completely. A525 must be taken quickly and consistently. 

Ultrasonic cell disrupters are manufactured by a half-dozen or so companies. In the author s lab, the Model 550 Sonic Dismenbrator (Fisher Scientific) has been in use. It becomes important to retune the generator when a new probe is changed. There are also additional tuning procedures to follow for microtips. 

For cell lysis, add 10 pL of lysostaphin solution (10 mg/mL) to the cell suspension. After incubation on ice for 10 min, disrupt cells by sonication. For this step, place a beaker of ice water around the sample tube to keep it cold. Sonicate the samples for 1 min (0.5/s, low) followed by a 1-min cooling break. Repeat this process three times. Sonication is complete when the solution appears noticeably less cloudy than the starting solution. 

Ultrasonication is another liquid-shear method of cell disruption. Ultrasonic vibrations having frequencies greater than 18 kHz are able to disrupt microbial cells in suspension. The ultrasonic vibration could be emitted continuously or in the form of short pulses. A frequency of 25 kHz is commonly used for cell disruption. The duration of this procedure depends on the cell type, the sample size and the cell concentration. The transmission of sonic waves creates a continuous cycle of microbubble cavitation in the suspending medium. These cavities or small bubbles of dissolved gases or... 

Disrupt cells by sonication (three bursts, 30 s each in a cold room) and remove debris by centrifugation (12,000x /15 min). Cells can also be gently lysed with lysozyme (2 mg/mL) at 4 °C before centrifugation. 

Sonication is a successful method which has been extensively used for cell disruption as a result of its speed, simplicity, cleanliness and capability to... 

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