RESUMO

A simple, reliable procedure for practically quantitative (90-98%) and fast (< 30 min) elution of proteins from SDS-PA gels is described with reproducible recoveries in the range from 100 to 1 pmol per band, which does not require the inclusion of detergents in the elution buffer. It consists in the combination of (1) highly sensitive on-gel protein detection (50 mol per band) with imidazole-SDS-zinc (reverse staining), (2) crushing of the protein band to produce 32-micron gel particles, and (3) vortexing of the slurry in a solution of a zinc-complexing agent, e.g. glycine 0.5 M or EDTA 100 mM (100 microliters for a 100-pmol BSA band), at room temperature. Eluted proteins can be directly analyzed by RP-HPLC, quantitatively loaded onto a PVDF membrane, or, provided that they are previously renatured on-gel, analyzed by biological activity tests. The application of the procedure to in-solution enrichment of scarce proteins for N-terminal analysis is shown.

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We present a new method for visualizing bacterial lipopolysaccharides (LPS)/lipooligosaccharides (LOS) electrophoresed in sodium dodecyl sulfate-polyacrylamide gels. After electrophoresis, gels are washed in boiling water to appreciably remove remaining electrophoresis reagents, then incubated in 10 mM zinc sulfate for 15 min, and subsequently immersed in 0.2 M imidazole for 3 min. As a result, zinc salts precipitate all over the gel surface except in the zones occupied by LPS/LOS, which appear as transparent, colorless bands. Gels can be stored in distilled water for weeks without loss of the negative image. The sensitivity of this stain is similar to that of silver. We believe that zinc-imidazole may be a suitable nontoxic alternative to silver in the rapid analysis of LPS/LOS by polyacrylamide gel electrophoresis.

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We developed a technique that allows rapid protein elution from polyacrylamide gel bands at room temperature into a detergent-free buffer (elution time 2 x 10 min, total working time about 30 min) with high yields (90-98%) even at a low picomole level (1 picomole per band). Its efficacy relies on the combination of protein detection by reverse staining with the enhancement of protein diffusion after gel crushing. Detection is accomplished by gel incubation in an imidazole solution, followed by incubation in a zinc salt solution to develop a negative stain pattern. Proteins are eluted by zinc complexation in Laemmli electrophoresis buffer (Tris + glycine), from which sodium dodecyl sulfate is omitted to allow direct subsequent microanalysis, e.g. high performance liquid chromatography (HPLC) and automatic sequencing. A variety of proteins were eluted efficiently (with no apparent restriction due to their intrinsic properties) as quantified with radioiodinated total E. coli proteins. Yields were independent of acrylamide concentration, protein molecular mass (from 10 to 100 kDa) and the amount (from 1 to 100 picomole) of protein in the band. This protocol was derived from a quantitative evaluation of the effect of protein staining and of sample reduction prior to electrophoresis on elution yields. For N-terminal sequencing, the protein eluate was automatically loaded on a polyvinylidene difluoride (PVDF) membrane with conventional HPLC equipment; both loading and membrane clean-up were monitored at 206 nm. By simultaneously processing several analytical bands, the procedure allowed trace enrichment of a natural scarce protein that was N-terminal sequenced.

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Assuntos

RESUMO

Nucleic acids separated by gel electrophoresis are commonly detected within the gel matrix with ethidium bromide staining, followed by gel irradiation with ultraviolet (UV) light. When the separated nucleic acids are to be recovered for further characterization or use, this methodology is unsuitable (i) because a significant number of chemical lesions to the nucleic acid molecules are caused, heavily compromising their biological activity, and (ii) because of health hazards due to accumulative direct contact with ethidium bromide and exposure to UV-light. As an alternative, for preparative purposes, a new nontoxic detection method employing zinc and imidazole salts is described. After electrophoresis, the gel is first washed in distilled water to substantially remove remaining electrophoresis reagents, then incubated in 40 mM zinc sulfate for 10 min to allow binding of Zn2+ to the DNA, and subsequently washed with distilled water to remove unbound Zn2+ from gel regions devoid of DNA. On soaking in 0.2 M imidazole for a few minutes, zinc-DNA complexes are visualized as deep-white (positive) stained bands against a slightly opaque background. The sensitivity is similar to that of ethidium bromide. Gels can be kept in distilled water for months without loss of staining. After zinc chelation, e.g. with EDTA, it is feasible to quantitatively recover chemically intact and biologically active DNA from the gels, as shown by reelectrophoresis and transformation experiments.

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Isolation of proteins from polyacrylamide electrophoresis gels by a novel combination of techniques is described. A given protein band from a reverse stained (imidazol-sodium dodecyl sulfate--zinc salts) gel can be directly electrotransferred onto a reversed-phase chromatographic support, packed in a self-made minicartridge (2 mm in thickness, 8 mm in internal diameter, made of inert polymeric materials). The minicartridge is then connected to a high-performance liquid chromatography system and the electrotransferred protein eluted by applying an acetonitrile gradient. Proteins elute in a small volume ( < 700 microL) of high-purity volatile solvents (water, trifluoroacetic acid, acetonitrile) and are free of contaminants (gel contaminants, salts, etc). Electrotransferred proteins were efficiently retained, e.g., up to 90% for radioiodinated alpha-lactalbumin, by the octadecyl matrix, and their recovery on elution from the minicartridge was in the range typical for this type of chromatographic support, e.g., 73% for alpha-lactalbumin. The technique was successfully applied to a variety of proteins in the molecular mass range 6-68 kDa, and with amounts between 50 and 2000 pmol. The good mechanical and chemical stability of the developed minicartridges, during electrotransfer and chromatography, allowed their repeated use. This new technique permitted a single-step separation of two proteins unresolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to their different elution from the reversed-phase support. The isolated proteins were amenable to analysis by N-terminal sequencing, enzymic digestion and mass spectrometry of their proteolytic fragments. Chromatographic elution of proteins from the reversed-phase mini-cartridge was apparently independent of the specific loading mode employed, i.e., loading by conventional loop injection or by electrotransfer.

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A methodology is presented for efficiently gaining structural information from electrophoresed proteins after on-gel detection by imidazole-sodium dodecyl sulfate-zinc reverse staining. As a consequence of reverse staining, (a) protein bands arise transparent against a deep white-stained background, limits of detection being in the femtomole range; (b) there is no loss of image when the gel is kept in distilled water (even during years); and (c) protein bands result immobilized, i.e., they do not diffuse upon gel storage. To recover reverse-stained proteins or fragments thereof from the gel, the immobilization of bands must first be abrogated by chelating the zinc ions from stain (protein mobilization). We had originally described mobilization at low pH by using citric acid. Here, we improve this procedure regarding the protein electrotransfer. We demonstrate that mobilization is efficiently done at neutral to alkaline pH by short-term (5 to 10 min) incubation of the gel in a buffer containing glycine or dithiothreitol prior to transfer. Moreover, mobilization was most simply performed by just adding the zinc chelating agent to the transfer buffer. Reverse staining and the new mobilization procedure made electrotransferring single protein bands from gel onto small-sized (13 x 5 mm2) PVDF membrane pieces in mini sandwich-like assemblies practical. Equipment is described for the protein electroblotting in such minisandwiches. Microsequence analysis of the electroblotted proteins showed initial yields in the range of those achieved when the transfer was done from unstained control gels. Protein bands kept in the reverse-stained gel for prolonged time periods (even for as long as 2 years) could be similarly analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)

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The sensitivity, simplicity, and relative rapidity of Coomassie blue staining have made this technique the method of choice for routine detection and quantitative analysis of gel electrophoresis-separated protein bands in many applications. To extend the usefulness of this technique, we have developed a new double-staining method for visualizing SDS-PAGE-separated protein bands that were undetected by Coomassie blue staining of the gel. Coomassie blue-stained gels are washed in distilled water (15 min, two times) and then subjected to imidazole-zinc reverse staining. As a result of the method, a homogeneous white-stained background is generated and two types of protein bands can be observed: (a) typical Coomassie blue-stained bands, which appear superposed on larger transparent bands; and (b) reverse-stained (transparent) bands, which were previously undetected by the Coomassie blue staining. The method is rapid, simple, and reproducible and double-staining gels can be kept in distilled water for months without loss of the protein pattern. The overall sensitivity is high (e.g., 1.6 ng for recombinant streptokinase, 47 kDa) over a wide range of protein molecular weights (10 to 100 kDa) and independent of the degree of Coomassie blue destaining of the gel. Furthermore, a mechanism offering a consistent explanation for the role of imidazole, SDS, and zinc in the reverse staining of gels, particularly after Coomassie blue staining is proposed.

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We report here a modification to copper and zinc chloride staining methods. The introduction of a preincubation of the gels, prior to metal staining, with 0.2 M imidazole allows the formation of a homogeneous background for the subsequent precipitation of the metal chelate. The reported imidazole-zinc staining takes minutes, resulting in reproducible staining patterns with only slightly lower sensitivity than silver staining. The method allows efficient recovery of proteins from previously stained gels and is compatible with immunoidentification on Western blots and also with amino acid analysis and NH2-terminal sequence analysis of transferred proteins. A mechanism is proposed to explain the observed improvement in reproducibility and sensitivity of imidazole preincubation to zinc staining.

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