Protein quantification and its tolerance for different interfering reagents using the BCA-method with regard to 2D SDS PAGE

Measuring the protein content of a sample is a mandatory and frequently practiced procedure in the lab. Although the procedure is quite simple and convenient to perform with commercially available kits, incompatible reagents in the lysate can cause problems in the quality of measurement. Unfortunately these reagents are cornerstones of high efficiency lysing buffers, e.g. high amounts of urea or beta-mercaptoethanol. In this study we addressed the tolerance of the well-known BCA-assay (bicinchoninic acid) to various reagents in different concentrations, with special regard to a subsequent 2D-gelelectrophoresis. As a result, the kit is incompatible with the recipes of regular 2D-buffers. Also, when mixing two different reagents interfering effects will occur in a non-predictable way. Therefore we established a new method to quantify protein content in lysates ready for 2D-gelelectrophoresis: by mixing an aliquot with SDS, an equilibration is performed to that the sample can be run on a regular 1D SDS PAGE. Image analysis following fluorescence staining (SYPRO Ruby) reveals the absolute protein content in comparison to a BSA dilution curve processed accordingly.     

Oligonucleotide labeling methods. 3. Direct labeling of oligonucleotides employing a novel, non-nucleosidic, 2-aminobutyl-1,3-propanediol backbone.

Novel CE-phosphoramidite (7a-e) and CPG (8a, c, d, e) reagents have been prepared from a unique 2-aminobutyl-1,3-propanediol backbone. The reagents have been used to directly label oligonucleotides with fluorescein, acridine, and biotin via automated DNA synthesis. The versatile 2-aminobutyl-1,3-propanediol backbone allows for labeling at any position (5', internal, and 3') during solid phase oligonucleotide synthesis. Multiple labels can be achieved by repetitive coupling cycles. Furthermore, the 3-carbon atom internucleotide phosphate distance is retained when inserted internally. Using this method, individual oligonucleotides possessing two and three different reporter molecules have been prepared.     

Synthesis of symmetrically and asymmetrically branched pegylating reagents.

A solution-phase procedure using an orthogonal protection scheme was developed for the synthesis of a novel family of multi-pegylating reagents. The procedure was exemplified by the synthesis of bis- and tris-pegylating reagents prepared by stepwise insertion of the poly(ethylene glycol) units thereby enabling the preparation of both symmetrical and asymmetrical pegylating reagents. Asymmetrical pegylation and tris-pegylation of peptides and proteins introduces new variables for use in the optimization of pegylated peptides and proteins. These reagents are ideally suited for conjugation to peptides and proteins as they possess a required functional group and will be useful intermediates for the synthesis of a new generation of pegylated products. Tris-pegylation can also provide more effective protection from proteolysis by shielding the protein surface from approaching macromolecules. To illustrate this potential, conditions were developed for the successful coupling of the tris-pegylating reagent to a model pentapeptide.     

Depth-dependent photolabelling of membrane hydrophobic core with 9-diazofluorene-2-butyric acid

Hydrophobic photoactivable reagents, which readily partition into membranes, have proved very useful for studying membrane hydrophobic core. These reagents have been linked to fatty acids in order to obtain amphipathic photoactivable reagents which label membranes more effectively. By varying the length of these amphipathic reagents, an attempt to label membrane hydrophobic core at different depths can be made. We report here 9-diazofluorene-2-butyric acid as a new photoactivable reagent which labels the single bilayer vesicles prepared from egg phosphatidylcholine. The labelling site on the fatty acyl chains could be traced to be between the carbon atom 4 and 6. The new probe thus labels the membrane at a site which is proximal to what can be predicted from its length and transverse location in membranes.     

A Simple Procedure for Crystallization of the Schiff Reagent

Formation of crystals in Schiff reagents prepared from SO₂ gas previously has been reported either soon after preparation, using high dye concentrations and heating, or after long periods of storage at room temperature. With the first type of procedure only a low yield of crystals accompanied by dye precipitation was obtained. Crystallization without dye precipitation took place if the reagent, prepared with pararosaniline base or chloride in a saturated SO₂ solution, was stored for a sufficient time at room temperature in partly filled flasks. These crystals remained colorless if washed with acid alcohol after being separated by filtration. Schiff reagents layered with paraffin oil or supplemented with 0.1 M hydroquinone took much longer to crystallize, suggesting that crystallizaticn is promoted by the partial oxidation of sulfurous acid to sulfuric acid. A high yield of crystals can be obtained at room temperature after as little as 24 hr by adding 0.04 M of H₂SO₄ to a Schiff reagent prepared with 2% pararosaniline chloride in a saturated SO₂ solution. A Schiff reagent prepared with only 0.2% of these crystals gives an intense staining in the Feulgen and in the Periodic acid-Schiff reactions.     

Determination of different amino sugar 2'-epimerase activities by coupling to N-acetylneuraminate synthesis.

A new procedure for quantitating the amount of N-acetyl-D-mannosamine (ManNAc) or ManNAc-6-phosphate produced by 2'-epimerase activities involved in sialic acid metabolism has been developed. The ManNAc generated by the action of N-acetyl-D-glucosamine (GlcNAc) and UDP-GlcNAc 2'-epimerases is condensed with pyruvate through the action of N-acetylneuraminate lyase and the sialic acid released is measured by the thiobarbituric acid assay. For the analysis of prokaryotic GlcNAc-6-phosphate 2'-epimerase, ManNAc-6-phosphate can also be evaluated by this coupled assay after dephosphorylation of the sugar phosphate. This system provides a sensitive, rapid, reproducible, specific and simple procedure (feasible with commercial reagents) for measuring amino sugar 2'-epimerases from eukaryotic and prokaryotic sources. The technique reported here permitted us to detect UDP-GlcNAc 2'-epimerase and GlcNAc 2'-epimerase in mammalian cell extracts and GlcNAc-6-phosphate 2'-epimerase in bacterial extracts.     

The Evaluation and Choice of Laboratory Equipment and Reagents

Many new types of laboratory equipment are appearing on the market, as well as a large number of reagent sets or kits. The importance of adequate evaluation of the reliability of such new products cannot be overemphasized.Commercially prepared reagent sets or kits fall into two categories. In the one, a set of reagents is offered for use in performance of a particular conventional procedure; this type of prepared reagent should present no problem, provided that the reagents are stable. In the other category, a reagent kit or set is offered which has been designed to simplify the performance of a laboratory analysis, often by combining reagents and reducing the number of steps and the time required. Such a kit should be evaluated carefully in order to determine its efficiency and limitations. In general it is difficult to simplify greatly a laboratory procedure without sacrificing its accuracy.     

Aspartate aminotransferase from chicken heart cytosol. Characterization of SH-groups]

Homogeneous aspartate aminotransferase has been prepared from chicken heart cytosol. The purification procedure includes fractionation with NH4-sulfate and with ethanol, chromatography on ion-exchange cellulose DE-32 and on hydroxylapatite. Crystallization of the enyme is described. The enzyme was shown to contain 4 SH-groups per protein subunit of molecular weight 50 000. Two of the SH-groups are fully buried, they can be blocked with thiol reagents only upon denaturation of the protein. One exposed SH-group is readily modified at alkaline pH by iodoacetamide, N-ethymaleimide or tetranitromethane, without any inhibition of enzymic activity; this group readily reacts also with 5,5,-ditthiobis (2-nitrobenzoate) and p-mercuribenzoate. One SH-group is semi-buried: it is inaccessible to the above-mentioned reagents at pH 8, but can be blocked by p-mercuribenzoate at pH about 5. Blocking with p-mercuribenzoate of two SH-groups-the exposed and the semi-buried one-lowers enzymic activity to 70% of the initial value. Syncatalytic modication of a SH-group observed in aspartate aminotransferase from pig heart cytosol does not occur in chicken enzyme.     

Substituted 2-iminothiolanes: reagents for the preparation of disulfide cross-linked conjugates with increased stability

Much attention has been focused recently on the stability of immunotoxin (antibody-toxin) conjugates linked by a disulfide bridge. Conflicting reports have appeared regarding the in vivo stability of such conjugates prepared with the two most commonly used cross-linking reagents, SPDP and 2-iminothiolane. We have developed (i) a series of reagents based on 2-iminothiolane substituted at the 4- and/or 5-positions (X2ITs) which, based on model studies with simple amines, should show enhanced disulfide stability when conjugated with antibodies or other proteins and (ii) a real-time method for monitoring the rate and extent of conjugation of these reagents with amino groups. Depending upon the substituent, the stability of model-activated disulfides relative to unsubstituted 2-iminothiolane was increased from 5- to 4000-fold as measured by glutathione-induced release of thionitrobenzoic acid. This family of cross-linking reagents should allow the construction of disulfide cross-linked toxin, drug, or enzyme conjugates with enhanced stability in vivo.     

Solid-phase synthesis of 2-[18F]fluoropropionyl peptides

The 2-[(18)F]fluoropropionic (2-[(18)F]FPA) acid is used as a prosthetic group for radiolabeling proteins and peptides for targeted imaging using positron emission tomography (PET). Radiolabeling of compounds with more than one acylable functional group can lead to complex mixtures of products; however, peptides can be labeled regioselectively on the solid phase. We investigated the use of a solid-phase approach for the preparation of 2-[(18)F]fluoropropionyl peptides. [(18)F]FPA was prepared and conjugated to the peptides attached to the solid phase support. The (18)F-labeled peptides were obtained in 175 min with decay corrected yields of 10% (related to [(18)F]fluoride) and with a purity of 76-99% prior HPLC purification. The suitability of various coupling reagents and solid supports were tested for radiolabeling of several peptides of various lengths.     

Automated solid-phase peptide synthesis: use of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate for coupling of tert-butyloxycarbonyl amino acids.

2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) has been adapted for use as a coupling reagent for tert-butyloxycarbonyl (Boc) amino acids in automated solid-phase peptide synthesis. When compared to the existing preformed symmetrical anhydride procedure employing dicyclohexyl-carbodiimide (DCC), the use of TBTU in the presence of 1-hydroxybenzotriazole (HOBt) provides a more efficient coupling procedure for Boc-amino acid derivatives. Overall cycle times using TBTU/HOBt coupling reagents (30 min) compare favorably to those of the DCC-mediated procedure (approx 65 min). Dimethylformamide can be used as the sole solvent for both activation and coupling reactions. Implementation of TBTU/HOBt coupling conditions does not require replumbing of any lines of the Applied Biosystems Model 430A instrument and necessitates changes to only three reagent bottle positions. The variable coupling efficiencies of Boc-asparagine following activation with TBTU/HOBt (as low as 89%) can be overcome by protection of the amide function of Boc-asparagine with the 9-xanthyl group. Examples of the synthesis and characterization of a number of peptides ranging in length from 13 to 29 residues are given.     

Synthesis of a cleavable protein-crosslinking reagent for the investigation of ribosome structure.

This communication describes a simple method for synthesizing cleavable bifunctional imido esters of different chain lengths. These reagents, which form covalent crosslinks between lysine residues of proteins, contain a disulfide bond which is cleaved under mild conditions by reducing agents such as 2-mercaptoethanol. The reagents are synthesized via the dithiobisnitrile which is prepared in high yield by reacting the appropriate omega-activated nitrile with sodium polysulfide and is then converted quantitatively to the diimidate. Three such reagents were prepared: dimethyl 3.3'-dithiobispropionimidate, dimethyl 4,4'-dithiobisbutyrimidate, and dimethyl 6-6'-dithiobiscaproimidate. The first was synthesized from acrylonitrile, and the others from the appropriate omega-bromonitriles. Experiments with the bispropionimidate and a test protein, pancreatic ribonuclease, have shown the reagent to be effective in producing multimeric crosslinked complexes, from which monomeric proteins can recovered after treatment with 2-mercaptoethanol. The reagents are suitable for studies of ribosomal structure.     

Qualitative determination of N-terminal amino acids of peptides and proteins with cobalt(III) chelates

1. A method of N-terminal peptide-bond hydrolysis with the cis-beta-hydroxyaquo(triethylenetetramine)cobalt(III) ion, i.e. beta-[Co(trien)(OH)(OH(2))](2+), is reported. The method has been demonstrated with 22 small peptides and ten proteins. 2. The procedure is rapid (an N-terminal amino acid determination can be made easily in one day), it involves no acid hydrolysis step and thus no destruction of labile amino acids, and it involves the use of easily prepared inexpensive reagents. 3. The released N-terminal amino acids can be identified as their cobalt(III) derivatives, or directly as the amino acid or as their dansylated derivatives. 4. The method is to treat 1 mumol of peptide or protein with beta-[Co(trien)(OH)(OH(2))](2+) reagent at pH8.0, 45 degrees C for 3h. Addition of 0.5m-phosphate buffer, pH10.5 at 45 degrees C for 10min cleaves the N-terminal bidentate amino acid-cobalt complex, which can be identified directly. For greater sensitivity with 10nmol of peptide) the free amino acid is prepared from the complex by treatment (with NaCN (0.1m, 40 degrees C, 30min), or H(2)S or NaBH(4) (25 degrees C, 5min), dried, dansylated and the dansyl-amino acid identified by high-voltage electrophoresis. The method is unaffected by the presence of 4-8m-urea, but will not cleave blocked N-terminal acids.     

Preparation and properties of fluorescent polysaccharides

A new method for preparing fluorescein derivatives of polysaccharides is described. These derivatives are prepared by activation of the polysaccharide with cyanogen bromide and subsequent reaction with fluoresceinamine. The optimum conditions for coupling have been established in this report. Using this procedure, we have prepared fluorescein derivatives of a wide variety of polysaccharides. Degrees of substitution in the range of 3.0 X 10(-3) to 2.4 X 10(-2) mol of fluorescein per mole of monosaccharide equivalent were obtained. The fluorescent derivatives are stable: no free fluorescein was detected after incubation at 22 degrees C for 48 h or at -10 degrees C for 4 months. The fluorescein-derivatized polysaccharides were found to have the same potency in inhibiting lectin-mediated hemagglutination as the underivatized polysaccharide. In addition, these fluorescent polysaccharides can be radioiodinated to specific activities exceeding 10(6) dpm/micrograms due to incorporation of 125I into fluorescein. The cell binding properties of 125I-fucoidin and 125I-heparin are indistinguishable from the corresponding underivatized polysaccharides. This general approach for preparing fluorescent polysaccharides should produce useful reagents for localizing and quantifying cell surface carbohydrate-binding proteins (lectins).     

N-(2-chlorobenzyloxycarbonyloxy)-succinimide as a terminating agent for solid-phase peptide synthesis: Application to a one-step purification procedure

Summary The solid-phase synthesis of peptides is limited by the ability to separate the target sequence from chromatographically similar deletion and truncated impurities. Earlier we have reported the development of a one-step purification procedure for Boc- and Fmoc-synthesised peptides, which involves the incorporation of a base-labile probe with enhanced chromatographic properties at the N-terminal residue of the peptidyl-resin. To prevent the coderivatisation of deletion peptides, an efficient capping procedure is required at each step of chain assembly to terminate unreacted amino groups. N-(2-Chlorobenzyloxycarbonyloxy)-succinimide (Z(2-Cl)-OSu) was found to be a highly effective capping agent for automated SPPS, because it is (i) a solid which can be dissolved when required to limit possible degradation; (ii) stable to the reagents commonly used for Boc/Fmoc chemistries; and (iii) sufficiently reactive so as not to significantly extend cycle times. We demonstrate the effectiveness of a 5 min capping cycle, using Z(2-Cl)-OSu, by synthesising several peptides ranging from 12 to 101 residues in length, by both the Fmoc and Boc chemical strategies.     

Solubilization and stabilization of bacteriophage MS2 in organic solvents

Several techniques were examined for the solubilization of bacteriophage MS2 in organic solvents. Direct extraction of the MS2 from an aqueous phase into isooctane containing 2 mM AOT, a proven approach for the organic solubilization of many proteins, was not successful. However, predried samples of MS2 were solubilized through the direct addition of organic solvents containing 500 mM AOT. As an alternative procedure, reverse micelles containing aqueous solutions of MS2 were prepared in isooctane using AOT, dehydrated through solvent evaporation and azeotropic drying, and resolubilized in a solvent of choice. The structure and microenvironment of organic-solubilized MS2 were investigated by UV absorbance, the fluorescence emission of an attached solvatochromatic dye, tryptophan fluorescence, and atomic force microscopy, all of which contributed evidence for a fully assembled capsid in the organic solvent. The solubilized MS2 was derivatized with stearic acid in chloroform, illustrating that bioconjugation reactions can be performed on organic-solubilized capsids using reagents that are completely insoluble in water. Furthermore, the organic-solubilized phage remained infectious after heating at 90 degrees C for 20 min, whereas phage in aqueous buffer or dried with nitrogen were nonviable following the heat treatment protocol. The extended range of available chemical modifications and the enhanced thermal stability of the organic-solubilized capsids bodes well for the formulation of storage-stable vaccines predicated on reactions in or exposure to organic media.     

Evaluation of a rapid membrane enzyme immunoassay (Testpack HIV-1/HIV-2) at zonal, regional and district hospital laboratories in Tanzania

The performance of a rapid and simple membrane enzyme immunoassay for antibodies to HIV-1 and HIV-2 (Testpack HIV-1/HIV-2) was evaluated by testing 1000 sera from the Kilimanjaro region of Tanzania. A sensitivity of 100% (118/118 positives) and specificity of 95.1% were obtained following the manufacturer's procedure. The specificity was significantly enhanced to 97.2% (P = 0.026) by modifying the Testpack procedure by including an extra was after serum adsorption to the unit membrane. The testing of a single specimen could be completed in 8 min and up to 10 individual tests could be run simultaneously. There was complete agreement in interpretation when the results were read independently by two trained technicians. A built-in control insured against incorrect procedures or inactive reagents. In a subsequent field trial including 450 sera, one strongly reactive sample failed to be detected at a participating field hospital for unknown reasons. The Testpack reagents proved stable for up to one year at room temperature (25-30 degrees C). The data indicate that Testpack is suitable for the detection of serum antibodies to HIV and is especially applicable in laboratories with limited facilities. When used to test African sera which are known to produce a high degree of false positivity, an extra wash of the membrane after serum adsorption is recommended.     

Development of an off-line capillary column IMAC phosphopeptide enrichment method for label-free phosphorylation relative quantification

Immobilized metal affinity chromatography (IMAC) and metal oxide type affinity chromatography (MOAC) techniques have been widely used for mass spectrometry-based phosphorylation analysis. Unlike MOAC techniques, IMAC requires rather complete removals of buffering reagents, salts and high concentrations of denaturant prior to sample loading in order for the successful enrichment of phosphopeptides. In this study, a simple off-line capillary column-based IMAC phosphopeptide enrichment method can shorten sample preparation time by eliminating the speed-vac step from the desalting process. Tryptic digest peptide samples containing 2M urea can be directly processed and the entire IMAC procedure can be completed within 6 h. When tryptic digest peptide samples prepared from mouse whole brain tissues were analyzed using our method, an average of 249 phosphoproteins and 463 unique phosphopeptides were identified from single 2-h RPLC-MS/MS analysis (~88% specificity). An additional advantage of this method is the significantly improved reproducibility of the phosphopeptide enrichment results. When four independent phosphopeptide enrichment experiments were carried out, the peak areas of phosphopeptides identified among four enrichment experiments were relatively similar (less than 16.2% relative standard dev.). Because of this increased reproducibility, relative phosphorylation quantification analysis of major phosphoproteins appears to be feasible without the need for stable isotope labeling techniques.     

An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications

Background

PCR amplification of minute quantities of degraded DNA for ancient DNA research, forensic analyses, wildlife studies and ultrasensitive diagnostics is often hampered by contamination problems. The extent of these problems is inversely related to DNA concentration and target fragment size and concern (i) sample contamination, (ii) laboratory surface contamination, (iii) carry-over contamination, and (iv) contamination of reagents.

Methodology/Principal Findings

Here we performed a quantitative evaluation of current decontamination methods for these last three sources of contamination, and developed a new procedure to eliminate contaminating DNA contained in PCR reagents. We observed that most current decontamination methods are either not efficient enough to degrade short contaminating DNA molecules, rendered inefficient by the reagents themselves, or interfere with the PCR when used at doses high enough to eliminate these molecules. We also show that efficient reagent decontamination can be achieved by using a combination of treatments adapted to different reagent categories. Our procedure involves γ- and UV-irradiation and treatment with a mutant recombinant heat-labile double-strand specific DNase from the Antarctic shrimp Pandalus borealis. Optimal performance of these treatments is achieved in narrow experimental conditions that have been precisely analyzed and defined herein.

Conclusions/Significance

There is not a single decontamination method valid for all possible contamination sources occurring in PCR reagents and in the molecular biology laboratory and most common decontamination methods are not efficient enough to decontaminate short DNA fragments of low concentration. We developed a versatile multistrategy decontamination procedure for PCR reagents. We demonstrate that this procedure allows efficient reagent decontamination while preserving the efficiency of PCR amplification of minute quantities of DNA.