Buffer, grinding

The result of 2D phoresis crucially depends on sample, sample treatment, and sample resolution. How do you extract (for example) the proteins from a liver sample Dissolve it directly in sample buffer Grind first in liquid nitrogen and then mix with sample buffer Lyophilize first and then dissolve in sample buffer No agreement has been reached yet. The next question is also unclear. What is the best sample buffer Because the first step of 2D phoresis is lEF, the proteins have to keep their own charge. That means you cannot dissolve with SDS. Nevertheless, the sample buffer has to dissolve as many proteins as possible and split them into subunits. Furthermore, it must prevent aggregation. Finally, the sample buffer should denature all proteases. Which sample buffer can do that None But some come close to this ideal (however, not very close). 

Resuspend in lysis buffer, freeze and grind under LN2... 

Procedure The Ascorbate peroxidase (EC 1.11.1.7) activity can be obtained by measuring the oxidation of ascorbate in the presence of H202. Grind the algal sample in liquid nitrogen and extract in 2.5 ml 50 mM potassium phosphate buffer (pH 7.0) containing 10% (w/v)... 

Preparations were made by grinding a sufficient amount of dry bacteria with phosphate buffer at pH 6.64 to result, after various further... 

Brumback [28] has described a custom robotic system designed to automate the extraction of proteins from plant samples. Leaf or callus material (5—25 mg) is presented to the robot in microcentrifuge tubes, the system performs buffer dispensing, grinding, centrifugation, and pipetting unit operations, and a cleared supernatant is delivered in a 96-well microassay plate format for subsequent analysis. 

Iin and co-workers [40] have developed a so-called micro-aqueous organic reaction system. In contrast to former preparations of almond meal, the almond kernels are soaked in water prior to grinding. After the defatting step, the meal contains 8-10% water (w/w), making it unnecessary to add the amount of water needed for enzyme activity. The reactions are carried out in buffer-saturated organic solvents to avoid a possible drying effect of the solvent on the biocatalyst. Further addition of water to the reaction results in lower conversions and values,... 

Early work was done with a sized fraction (about 0.1 mm) obtained by grinding zeolite pellets which contained a significant amount of binder. Subsequently, the pure zeolite powder was used. In all cases the zeolite was washed with a large volume of dilute salt solution, sometimes containing a small amount of acetate buffer at about pH 5.5, and precautions were taken to avoid hydrolytic precipitation of the metals. 

The isolation procedure starts with the preparation of a cell extract in which the enzyme activity can be detected. This typically involves grinding the tissue in an extraction buffer so that the cell contents, including the proteins, become accessible. Protease inhibitors, such as phenylmethyl... 

Turning to assays using sensors that require more than a simple cut in the sample, Bergann et al. [37] paid considerable attention to sample preparation in attempts to measure lactate in meat with a sensor (not thick film) based on reaction of hydrogen peroxide with a platinum electrode or on the reaction of ferrocene carboxylate with the active site of lactate oxidase. Sample preparation entailed extraction into buffer following grinding. In some cases, ground samples were left to allow the lactate to diffuse into the buffer solution. This was quite effective but slow (up to 90 min). Ultimately, the quality of the assay was dependent on the method of sample preparation. 

Grind infected material (see Note 9) in a muslin-lined bag in 0.06 M phosphate buffer, pH 6.5, with a hand grinder (see Note 10) to a dilution of 1 10. 

A sensitive reverse-phase HPLC method has been developed for the analysis of etodolac in tablet formulation [22]. The chromatographic separation was achieved using a reverse-phase Cu column, having dimensions of 3.3 cm x 0.46 cm i.d. (3 pm particles) and which was maintained at 30°C. The mobile phase consisted of pH 6.0 phosphate buffer / methanol (60 40 v/v), and was eluted at 1 mL/min. Analyte detection was effected on the basis of UV detection at 230 nm. Diazepam was used as an internal standard. The sample preparation entailed grinding the etodolac tablets, followed by extraction with methanol (using sonication). A retention time of 1.46 min was obtained for etodolac under these conditions, and the method was found to be linear, precise, and accurate over the concentration range of 0.01 to 0.1 mg/mL. 

Swell the dry beads made of cross-linked dextran, polyacrylamide or gelatin in PBS-A or Hepes buffered saline as described by the manufacturer. The cross-linked dextran beads require 2-3 h to swell at room temperature. Avoid stirring with a simple bar magnet as this may grind the beads. Addition of non-ionic detergent (e.g. Tween 80) to 0.1% may help initial wetting of the microcarrier. 

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