Preparation of Escherichia coli elongation factor Tu-guanosine 5'-triphosphate analogs

A simple procedure for the bulk preparation of 20 mg of Escherichia coli elongation factor (EF)-Tu-GTP analogs is described. The protocol is based upon the preparation and stabilization of nucleotide-free EF-Tu using an EF-Ts affinity chromatographic resin. The procedure is a general one for the preparation of any GTP analog of EF-Tu.     

Isolation of human erythrocyte membranes in glucose solution

A method is described for the preparation or removal of erythrocyte membranes from hemolysates by a glucose solution. The procedure is simple and rapid, requiring centrifugation at 8000g for 2 min. The preparation has microscopic shape and two-dimensional peptide patterns similar to those of the membrane isolated by conventional procedures (10,000g for 20 min). The present procedure is suitable for dealing with a bulky preparation or for removal of erythrocyte membranes from large volumes of hemolysates to purify enzymes and proteins of soluble or membrane fractions.     

Biochemical and structural characterization of actin from Dictyostelium discoideum.

Actin has been purified from amoebae of Dictyostelium discoideum by a procedure which is notable in that proteolysis has been diminished to undetectable levels and "selective" purification steps have been avoided. The overall yield of this procedure is 5- to 10- fold greater than that of a previous report (Spudich, J. A. (1974) J. Biol. Chem. 249, 6013-6020). The detailed biochemical and structural properties of this new preparation (preparation B) have been compared to those of Dictyostelium actin prepared by the previous procedure (preparation A) as well as to rabbit skeletal muscle actin. Preparation B actin is similar to muscle actin in its molecular weight, ability to activate myosin, filament structure, and polymerization properties. Preparation B actin has the same molecular weight and isoelectric point as preparation A actin, which is more acidic than that of skeletal muscle actin. However, preparation B actin and muscle actin form longer filaments than preparation A actin, as judged by viscometry and electron microscopy.     

Improved sample preparation for the testosterone hydroxylation assay using disposable extraction columns

The preparation of samples for injection into a high-performance liquid chromatography from assay mixtures for the determination of cytochrome P-450-dependent testosterone hydroxylation has been substantially facilitated. By replacing the multiple cumbersome extraction steps of the conventional method with a single column extraction the time for sample preparation was reduced from hours to minutes. The new procedure also yields better recoveries for most of the testosterone metabolites than the original protocol. The use of extraction columns for sample preparation allows the simultaneous treatment of a large number of samples or even the automation of the whole assay procedure. The modified procedure is a straightforward, easy-to-perform method that should greatly facilitate the implementation of the testosterone hydroxylation assay for sharply discriminating between many individual cytochrome P-450 species in routine enzyme diagnostics.     

Semi-automated liquid--liquid back-extraction in a 96-well format to decrease sample preparation time for the determination of dextromethorphan and dextrorphan in human plasma

A semi-automated, 96-well based liquid-liquid back-extraction (LLE) procedure was developed and used for sample preparation of dextromethorphan (DEX), an active ingredient in many over-the-counter cough formulations, and dextrorphan (DOR), an active metabolite of DEX, in human plasma. The plasma extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The analytes were isolated from human plasma using an initial ether extraction, followed by a back extraction from the ether into a small volume of acidified water. The acidified water isolated from the back extraction was analyzed directly by LC-MS-MS, eliminating the need for a dry down step. A liquid handling system was utilized for all aspects of liquid transfers during the LLE procedure including the transfer of samples from individual tubes into a 96-well format, preparation of standards, addition of internal standard and the addition and transfer of the extraction solvents. The semi-automated, 96-well based LLE procedure reduced sample preparation time by a factor of four versus a comparable manually performed LLE procedure.     

A Rapid, High-Yield Purification of L-Alanine:4,5-Dioxovalerate Transaminase from Rat Kidney Mitochondria Using an Improved Enzyme Assay Method

The present report documents an improved enzyme assay method for the mammalian L-alanine:4,5-dioxovalerate transaminase which is of significant utility in work with crude tissue homogenates, cell cultures, or purified enzyme preparations. We also describe a new and rapid purification procedure for this enzyme from rat kidney mitochondria. The three-step procedure involves the use of digitonin and lubrol for mitochondrial matrix preparation and L-alanine-Sepharose 4B column chromatography followed by gel filtration on Sepharose 6B. By this procedure it is possible to obtain a highly purified enzyme preparation in a relatively short time with a 37.5% yield.     

Preparation of radioactive aldehyde-containing phosphatidylcholine

Short-chain, aldehyde-containing phosphatidylcholine (PC), formed during the oxidation of PC, is thought to be involved in cellular responses in atherosclerosis and inflammation. Here we report a convenient procedure for a small-scale preparation of aldehyde-containing PC. PC containing an unsaturated fatty acyl chain was treated with osmium tetroxide followed by sodium periodate at room temperature. The reaction product was purified by TLC. This preparation showed a single peak on reverse-phase HPLC, and its identity was confirmed by fast atom bombardment mass spectrometry. This procedure does not require special equipment and is easily applicable for preparation of radioactive materials.     

A comparison of alternative sample preparation procedures for the analysis of swainsonine using LC‐MS/MS

Introduction – Swainsonine, a polyhydroxy indolizidine alkaloid and known glycosidase inhibitor, is found in a number of different plants that cause a lysosomal storage disease known as locoism in the western USA. Most recently swainsonine has been analysed by LC‐MS/MS after sample extraction and preparation from ion‐exchange resins. Objective – To compare previously published sample preparation procedures with several new alternative procedures to provide methods using either commercially available solid‐phase extraction equipment or procedures which significantly reduce sample preparation time. Methodology – A previously reported and validated sample preparation method using ion‐exchange resin was compared with methods using a commercially available solid‐phase extraction cartridge, a solvent partitioning procedure or a single solvent extraction procedure using one of two solvents. Twenty different plant samples of varying swainsonine concentrations were prepared in triplicate and analysed by LC‐MS/MS. The measured concentration of swainsonine was then statistically compared between methods. Results – There were no statistically significant differences found between four of the five different sample preparation methods tested. Conclusion – A commercially available SPE cartridge can be used to replace the previously used ion‐exchange resin for swainsonine analysis. For very rapid analyses the SPE procedure can be eliminated and a simple, single solvent extraction step used for sample preparation. Published in 2010 by John Wiley & Sons, Ltd.     

Sample preparation for two-dimensional blue native/SDS polyacrylamide gel electrophoresis in the identification of Streptomyces coelicolor cytoplasmic protein complexes

Ammonium sulfate precipitation was tested as a sample preparation step for BN-PAGE analyses of S. coelicolor cytoplasmic protein complexes. A procedure of sample preparation compatible with two-dimensional BN/SDS-PAGE was established and used to visualize protein complexes. To validate the sample preparation procedure, representative protein complexes were identified. Several previously characterized protein complexes were rediscovered and their reported oligomeric states reconfirmed. In addition, we identified new but plausible interactions that have never been reported before. Our work provides useful reference for the wide application of BN-PAGE in protein interaction study.     

A simple procedure to obtain plasmids suitable for transfecting mammalian cell lines

A simple procedure to obtain plasmid preparations, suitable for transfecting mammalian cell lines using a calcium phosphate co-precipitation technique, is described. The protocol is based on the purification of plasmid DNA by double gel-filtration chromatography on Sephacryl S-1000 and additional slight modifications to the original transfection procedure. The purity of plasmid preparation was verified by analytical methods. The resulting preparation efficiently transfected NIH-3T3 cells.The authors are with the National Center for Scientific Research, Molecular Biology Department, Biotechnology Branch, POB 6880, Havana, Cuba.     

Simple and rapid purification of brevin

Brevin or plasma gelsolin, a calcium dependent actin-binding and actin-severing protein, was purified from bovine plasma by a very rapid and simple procedure; ammonium sulfate fractionation and only one step of anion exchange column chromatography by a convenient use. It takes only 24 hrs to complete all the procedure. The purity of brevin prepared by this method was more than 95% on SDS-PAGE and total recovery was much better than previous preparation methods. This brevin preparation has about 8 isomers on 2-D PAGE and strong severing activity on F-actin under electron microscopic observation.     

An optimized PCR-based procedure for production of 13C/15N-labeled DNA

We have substantially improved a procedure that we previously described for producing 13C/15N-labeled DNA (Chen et al., FEBS Lett. 436, 372-376, 1998) to provide an economical and straightforward approach to the preparation of labeled DNA. The conditions for the PCR reactions have been optimized to permit the use of low concentrations of the costly labeled dNTPs (50 microM for each). In addition, a rapid and high-yield purification procedure has been developed that allows us to obtain a high yield of very pure labeled DNA. These modifications to our original procedure permit us to obtain 1.9 mg of an 18 bp DNA oligomer from 20 mg of dNTPs (ca. 10% yield from the starting dNTPs). This is sufficient material for the preparation of 0.4 mM sample in a volume of 400 microl. In summary, this procedure is a cost-effective, time-efficient procedure for the production of labeled DNA for NMR studies.     

Sample preparation for two-dimensional blue native/SDS polyacrylamide gel electrophoresis in the identification of Streptomyces coelicolor cytoplasmic protein complexes

Ammonium sulfate precipitation was tested as a sample preparation step for BN-PAGE analyses of S. coelicolor cytoplasmic protein complexes. A procedure of sample preparation compatible with two-dimensional BN/SDS-PAGE was established and used to visualize protein complexes. To validate the sample preparation procedure, representative protein complexes were identified. Several previously characterized protein complexes were rediscovered and their reported oligomeric states reconfirmed. In addition, we identified new but plausible interactions that have never been reported before. Our work provides useful reference for the wide application of BN-PAGE in protein interaction study.     

Isolation of three electrophoretic variants of rat pituitary growth hormone

A procedure has been developed for the isolation of rat pituitary growth hormone and for the subsequent resolution of the preparation into three variants by preparative electrophoresis. The starting material was whole frozen glands and the process involved homogenization and extraction at pH 6.2, ammonium sulfate fractionation and molecular-sieve chromatography on Sephadex G-100. The separation into charge variants was achieved by zone electrophoresis in agarose suspension at alkaline pH. The purification was monitored by radioimmunoassay and the specific activities were expressed in terms of the rat growth hormone reference preparation (RP-1) supplied by the NIADDK, Bethesda, U.S.A. The three-component preparation and its constituents all had activities in the same range, exceeding the activity of the reference by a factor up to 20 times. Bioassay of the three-component preparation, based on measurement of longitudinal bone growth in hypophysectomized rats gave a potency of 4-5 IU/mg. The reference was the 1st International Standard (bovine) for growth hormone. The yield of the three-component preparation was 3.3 mg per gram pituitary tissue. Different electrophoretic analyses revealed the efficiency of the preparative procedure in separating the variants. The results of the analyses also support the view that difference in electrophoretic behaviour is due to a difference of a single net charge between adjacent variants. In addition, growth hormone was prepared from two side extracts (at pH 7.0 and pH 9.8, respectively), provided by a procedure developed earlier for rat prolactin. The three preparations gave electrophoretic patterns of equal appearance although the relative proportions of the activity peaks differed.     

Isolation of Three Electrophoretic Variants of Rat Pituitary Growth Hormone

A procedure has been developed for the isolation of rat pituitary growth hormone and for the subsequent resolution of the preparation into three variants by preparative electrophoresis. The starting material was whole frozen glands and the process involved homogenization and extraction at pH 6.2, ammonium sulfate fractionation and molecular-sieve chromatography on Sephadex G-100. The separation into charge variants was achieved by zone electrophoresis in agarose suspension at alkaline pH.

The purfication was monitored by radioimmunoassay and the specific activities were expressed in terms of the rat growth hormone reference preparation (RP-1) supplied by the NIADDK, Bethesda, U. S. A.

The three-component preparation and its constituents all had activities in the same range, exceeding the activity of the reference by a factor up to 20 times. Bioassay of the three-component preparation, based on measurement of longitudinal bone growth in hypophysectomized rats gave a potency of 4–5 IU/mg. The reference was the 1st International Standard (bovine) for growth hormone. The yield of the three-component preparation was 3.3 mg per gram pituitary tissue.

Different electrophoretic analyses revealed the efficiency of the preparative procedure in separating the variants. The results of the analyses also support the view that difference in electrophoretic behaviour is due to a difference of a single net charge between adjacent variants.

In addition, growth hormone was prepared from two side extracts (at pH 7.0 and pH 9.8, respectively), provided by a procedure developed earlier for rat prolactin. The three preparations gave electrophoretic patterns of equal appearance although the relative proportions of the activity peaks differed.     

Purification of Cytochrome b5 from Pig Testis Microsomes by Isoelectric Focusing in an Immobiline pH Gradient

Testicular cytochrome b5 was purified by a procedure including preparative isoelectrofocusing. The cytochrome b5 was determined to have an isoelectric point of 4.45 on analytical isoelectric focusing. The purified cytochrome b5 was found to be homogeneous and its molecular weight was estimated to be 16,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The oxidized and reduced forms of the purified preparation exhibited absorption spectra of a typical cytochrome b5. A 69-fold purification was achieved with an overall yield of 6.2%. Following preparation of the microsomes, the purification is accomplished by a two-step procedure utilizing column chromatography and preparative isoelectric focusing.     

Improved large scale purification procedure of natural human fibroblast interferon

Human fibroblast interferon, produced in tissue culture by a superinduction procedure, was purified 10,000-fold by chromatography to ca 50% purity as judged by SDS-PAGE. The specific activity of the interferon preparation is 2 X 10(8) and the yield is 70%. The interferon preparation is suitable for clinical use.     

Rapid purification of plasmid DNAs by hydroxyapatite chromatography.

A method is described for the rapid preparation of plasmid DNAs of molecular weight up to 14 X 10(6). This method involves the chromatography, at room temperature, of bacterial cleared lysates on hydroxyapatite in the presence of high concentrations of phosphate and urea. All detectable protein and RNA contamination of plasmid DNA is removed by this procedure and the conformation of the plasmid DNA is unaffected. Less than 0.5% chromosomal DNA is present in the purified preparation and even this can be removed if necessary by a simple extention of the procedure to include a heat-denaturation step. The method is extremely rapid and amenable to large-scale plasmid preparation; 5 mg ColE1 DNA have been purified within 40 min. The yield of plasmid DNA is similar to that obtained with the conventional dye-centrifugation technique, however the purity is greater.     

Novel sample preparation method facilitating identification of urinary drug metabolites by liquid chromatography–tandem mass spectrometry

A simple, efficient procedure was developed for the preparation of urine samples, which greatly facilitated the identification of the urinary metabolites of a new antifungal agent SYN-2836. The urine samples following dilution with acetonitrile (ACN) formed distinct upper (ACN) and lower (aqueous) solution phases. The SYN-2836 metabolites were concentrated in the upper solution except that two glucuronides were concentrated in the lower solution. The upper solutions, containing concentrated metabolites and significantly reduced endogenous polar species, were ideally suitable for the metabolite identification. This novel sample preparation procedure would be applicable in identification of urinary metabolites of other drugs and drug candidates.     

Proteolytic degradation of ferredoxin-NADP reductase during purification from spinach

Ferredoxin-NADP reductase (FNR) was rapidly isolated from spinach leaves with special care to suppress proteolytic degradation. The molecular mass of this FNR preparation was estimated to be 35 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Limited proteolysis of 35-kDa FNR to 33-kDa FNR was effectively suppressed by high pH (at pH 9.3), concentrated salts, and low temperature. On the basis of these observations, a new isolation procedure was designed to obtain 35-kDa FNR in a preparative scale. The resulting final preparation still contained two FNR components. One appeared to correspond to the longest polypeptide so far reported for spinach FNR (Karplus et al., 1984, Biochemistry 23, 6576-6583) while the other lacked a gamma-pyroglutamyl residue from its amino terminus. Conventional preparation procedure without suppression of proteolytic action yielded an FNR preparation with a molecular mass of 33 kDa. This FNR preparation consisted of three components. They lacked 11 to 17 amino-terminal residues, while their carboxyl-terminal structure was retained intact. These results showed that proteolytic degradation of the spinach FNR molecule during purification took place exclusively at its amino-terminal moiety and further suggested that 35-kDa FNR with Karplus' structure should be the mature FNR molecule functional in the chloroplast thylakoids.