Preparation of protein samples

If precast gels are provided, move ahead to the preparation of protein samples for electrophoresis. If gels are to be made, follow these instructions for preparation of an SDS gel of 12% acrylamide. 

There are two key aspects to the preparation of protein samples for measurement by solid-state NMR. The hrst is the chemical or biochemical process by which the protein molecules are produced, with particular emphasis on the inclusion or exclusion of particular nuclear isotopes. The second is the means by which the protein molecules are prepared for the actual measurement, with possibilities including lyophilization, crystallization and insertion into lipid bilayers or liposomes. 

Gel filtration is a rapid and efficient alternative to dialysis for desalting and buffer exchange. It is used, for example, in the preparation of protein samples prior to freeze drying or fractionating by ion exchange chromatography. Applications include ... 

The present emphasis is put on general chemistry testing and immtmoassays based on absorption, agglutination, or fluorescence detection. Also the preparation of protein samples for subsequent analysis by MALDI-MS has been... 

Microfluidic devices are used in a variety of profiling and functional proteomics applications the goal in much of this work is to reduce sample and reagent consumption and increase throughput. The primary application in profiling proteomics is in the preparation of protein samples for separations and mass spectrometry, while the major functional proteomics tools are enzymatic and immxmological assays. 

Cell lysis When studying signaling components, it is important that the integrity and phosphorylation states of proteins of interest are not altered during cell lysis and the subsequent preparation of the samples prior to analysis. The principal precautions include the use of inhibitors of protein phosphatases and of proteases, in addition to working speedily and keeping samples cold (0 to 4°). 

Until recently, cell-free protein expression (also sometimes erroneously named in vitro protein expression) did not exhibit the productivity required for preparation of NMR samples, especially considering the high cost of using isotopically labeled starting material. Rather, it was exclusively used as an analytical tool that served to verify correct cloning or to study promotor sites. Because of the very low yields, detection of the expressed product usually required incorporation of a radioactive label (usually via 35S-methionine). 

The preparation of the sample prior to its analysis will depend upon the nature of both the sample and the analytical method chosen and may involve the disruption of cells, homogenization and extraction procedures as well as the removal of protein or other interfering substances. It may be necessary to prevent the decomposition and degradation of the carbohydrate content during such treatments or during storage by the addition of antibacterial agents such as thymol or merthiolate, or substances such as fluoride ions, which will inhibit the enzymic transformation of the carbohydrates. 

Mix all the components well. Add 8 fiL of pure TEMED. Mix well and immediately pour between glass plates. Fill the space to the top of the plates. Immediately insert the comb to make sample wells in the gel. Allow the gel to polymerize (about 30 min). Remove the comb and carefully rinse the sample wells with a 1 10 dilution of Tris-glycine SDS buffer. Prepare each protein sample including the molecular weight standards as follows ... 

Perform enzymatic hydrolysis of protein samples at a series of time points (see Basic Protocol 1, steps 1 to 5). Prepare a series of standards and a blank (see Basic Protocol... 

Novobiocin (NBC) is used for the treatment of mastitis in dairy cattle. In 1982, the tolerance level was set at 100 pg/kg in milk from dairy animals. An HPLC assay was developed for NBC determination in bovine milk at a tolerance level (214). The milk sample was diluted with buffer, the proteins were precipitated with MeOH, and the solution was filtered. The filtrate was injected directly into an HPLC system with UV detection. The interlaboratory study was realized for the analysis of two concentrations of NBC in fortified control milk samples. Recoveries of NBC reported by the participating laboratories were 89-99% at 50 pg/kg, 93-101% at 100 pg/kg, and 89-100% at 2 mg/kg. The CVs ranged from 2.0% to 6.2%. All laboratories described the procedure as rapid and simple, allowing the preparation of 20 samples in less than 2 hours. 

Narkis and Henfield-Furie [578] have described a direct method for the identification and determination of volatile water-soluble Ci C5 acids in municipal waste water and raw sewage. The method involves direct injection of the sewage into a gas chromatograph equipped with a Carbowax 20m on acid-washed Chromosorb W column and a flame ionisation detector. Preliminary preparation of the sample is limited to the addition of solid metaphosphoric acid to the sewage and removal of precipitated proteins and suspended solids by centrifuging. 

As a rule, a separation method should be used for both purification and concentration of the sample. The classic method for peptides and proteins is a reverse-phase liquid chromatography preparation of the sample, followed by a concentration step (often lyophiliza-tion) of the fraction of interest. During those steps performed on very small quantities of sample, loss on the sample can occur if care is not taken to avoid it. Lyophilization, for instance, can lead to the loss of the sample absorbed on the walls of the vial. The use of separation methods on-line with the mass spectrometer often are preferred. Micro- or nano-HPLC [32,33] and capillary electrophoresis [34], both coupled mainly to electrospray ionization/mass spectrometry (ESI-MS), are used more and more. 

Figure 4.9 The preparation of a sample from a reaction mixture that contains an excess amount of protein prior to injection and analysis by HPLC. (A) Termination carried out by immersion of reaction sample in a sand bath maintained at 1SS°C (B) Removal of the precipitated protein by either centrifugation or filtration. (C) Injection and analysis of the clarified solution.

The enzymatic assay is then described, including buffers and pH, the method for initiating the reaction, and the process used for termination. Next, the methods used in the preparation of the sample for HPLC analysis are described, including centrifugation, filtration, or any type of purification preceding injection into the HPLC system. For many of the assays, time span and range of protein concentration for which the reaction is linear are also indicated. 

Reaction with DFP. Chymotrypsin preparations oxidized with NBS to produce varying degrees of inactivation were treated with DFP and their phosphorus content then determined. An enzyme sample which had lost more than 60 % of its original activity was still able to incorporate 1 mole of phosphorus (DIP-group) per mole of protein. Samples with enzymatic activity equal to 15 % of the initial value or less incorporated 0.6 mole of phosphorus per mole of protein. The data on the extent of phosphorus... 

This cross-axis CPC provides the universal application of protein samples with a dextran-PEG polymer-phase system. Using a prototype of the L cross-axis CPC with a 130-mL column capacity, profilin-actin complex was purified directly from a crude extract otAcanthamoeba with the same solvent system as used for the serum protein separation earlier. The sample solution was prepared by adding proper amounts of PEG 8000 and dex-tran T500 to 2.5 g of the Acanthamoeba crude extract to adjust the two-phase composition similar to that of the solvent system used for the separation. The experiment was performed by eluting the upper phase at 0.5 mL/min under a high revolution rate of 1000 rpm. The profihn-actin complex was eluted between 60 mL and 84 mL fractions and well separated from other compounds. The retention of the stationary phase was 69.0% of the total column capacity. 

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