Micropellicular, anion-exchange column packings are used in chromatofocusing to demonstrate the resolution and speed achieved when proteins are separated under these conditions. Linear or concave pH gradients are produced with simple mixtures containing four or fewer individual buffering species instead of the more commonly used polyampholyte buffers. Computer-aided design methods are demonstrated for selecting the composition of the elution buffer to produce a pH gradient of a desired shape.
The method is applied to high-resolution, analytical- and preparative-scale separations involving horse myoglobin, human hemoglobin variants, and bovine carbonic anhydrase. A useful selection of buffering species is described capable of producing pH gradients of a variety of shapes in the range between pH 9.5 and 5.5.
Enzyme-linked immunosorbent assay-based selection and optimization of elution buffer for TAG72-affinity chromatography
An enzyme-linked immunosorbent assay (ELISA)-elution assay was developed to screen a large variety of elution buffers for selection of a suitable one for purification of the fusion protein FV/TNF-alpha by affinity chromatography. Various commonly used buffer systems utilizing widely differing conditions such as extreme pH, denaturants, chaotropic ions and polarity reducing reagents were investigated. Ammonia solution (1 M, pH 11.5) proved to exert the most suitable influence on dissociation of the FV/TNF-alpha/TAG72 complex while having a minimal protein denaturing effect on FV/TNF-alpha. The total yield of purified FV/TNF-alpha using the TAG72-affinity column with this elution system was 300-fold higher than that using the common elution buffer, 0.1 M glycine, 0.5 M NaCl, pH 2.7. Our study indicates that the ELISA-elution assay will be most useful in the selection of suitable elution buffers for affinity chromatography.
Effect of three elution buffers on the recovery and structure of monoclonal antibodies.
Antibodies are routinely purified by acid/salt elution from antigen affinity columns. The antibodies recovered with this procedure are active, but the recovery of protein is often low. We investigated the effect of acid and other denaturing or chaotropic solvents on the conformation of monoclonal antibodies (mAbs) made against the extracellular region of Her2 receptor (sHer2) derived from Chinese hamster ovary cells. The mAb remain almost completely folded in the 0.1 M glycine, pH 2.9, commonly used for elution, with the beta-sheet secondary structure intact, and only very small changes detected in the environment of the tryptophans.
In 7 M urea, 50 mM NaAc pH 4.0, the antibody was partially unfolded, with the Trp environment further perturbed and some of the beta-sheet structure converted to disordered structure. In 6 M guanidine HCl, 50 mM NaAc, pH 4.0, the antibody is completely unfolded, with no secondary or tertiary structure present. The antibodies exposed to glycine or urea were refolded by dialysis into phosphate-buffered saline (PBS), while the guanidine HCl-denatured antibodies were refolded by dialysis into 7 M urea, pH 4.0, followed by dialysis into PBS.
The refolded antibodies were capable of forming antigen-antibody complexes which could be isolated by gel filtration chromatography. Two different mAbs were subjected to immunoaffinity chromatography on sHer2-Sepharose. mAb86 was eluted by 0.1 M Gly, pH 2.9, while mAb52 was eluted with the 7 M urea, 50 mM NaAc, pH 4.0. The isolated antibodies were refolded by dialysis into PBS, analyzed for their ability to recognize native sHer2 by immunoprecipitation, and denatured sHer2 by Western blot analysis. Both preparations recognized the native protein, but precipitated slightly different forms of sHer2, indicating that they might recognize different epitopes. The mAb52 is a more sensitive reagent for Western blot analysis. Thus, this procedure can be used to recover antibodies which would not be recovered with glycine as the only eluate. It is also possible that the antibodies can be fractionated by the different eluants into populations which can be used for different applications.
Elution and reconcentration of coliphages in water from positively charged membrane filters with urea-arginine phosphate buffer
Coliphages in drinking water and waste water samples have been adsorbed onto positively charged membrane filters, eluted with urea-arginine phosphate buffer (UAPB), reconcentrated, and detected with Escherichia coli C (ATCC 13706). The proposed membrane filter-based UAPB method for concentration and detection of coliphages compares favorably with the beef extract elution and reconcentration procedure and also with the proposed coliphage detection procedure described in Standard Methods for the Examination of Water and Wastewater. The higher recovery of coliphages with UAPB elution from positively charged membrane filters is attributed to testing the whole volume of concentrated sample, rather than partial analysis of the sample as in the procedure described in Standard Methods for the Examination of Water and Wastewater, especially when the titre is very low.
Effect of buffer solution pH on the elution and separation of beta-blockers by micellar electrokinetic capillary chromatography
Study was made of the effect of the pH of phosphate buffer (0.08 M) containing 15 mM cetyltrimethylammonium bromide as surfactant on the elution order of eleven widely used beta-adrenergic blocking agents. In the pH range 6.0-7.8 the elution order of six of the beta-blockers remained the same, while the order of five of them changed. Sotalol eluted as the sixth compound at pH 6.8 and migrated more quickly with increasing pH. Below pH 7.0 labetalol eluted before propranolol and above pH 7.0 afterwards. Likewise, the order of elution of atenolol and timolol was reversed at pH 7.0. The pH also affected the resolution; the best resolution values were achieved between pH 6.6 and 7.0 and between pH 7.4 and 7.8. The relationship between the structure of the beta-blockers described by molecular and molecular connectivity indices and the elution order and separation of the beta-blockers in micellar electrokinetic capillary chromatography at varying pH of the buffer solution is discussed.
DNA adsorption to and elution from silica surfaces: influence of amino acid buffers
Solid phase extraction and purification of DNA from complex samples typically requires chaotropic salts that can inhibit downstream polymerase amplification if carried into the elution buffer. Amino acid buffers may serve as a more compatible alternative for modulating the interaction between DNA and silica surfaces. We characterized DNA binding to silica surfaces, facilitated by representative amino acid buffers, and the subsequent elution of DNA from the silica surfaces. Through bulk depletion experiments, we found that more DNA adsorbs to silica particles out of positively compared to negatively charged amino acid buffers.
Additionally, the type of the silica surface greatly influences the amount of DNA adsorbed and the final elution yield. Quartz crystal microbalance experiments with dissipation monitoring (QCM-D) revealed multiphasic DNA adsorption out of stronger adsorbing conditions such as arginine, glycine, and glutamine, with DNA more rigidly bound during the early stages of the adsorption process. The DNA film adsorbed out of glutamate was more flexible and uniform throughout the adsorption process. QCM-D characterization of DNA elution from the silica surface indicates an uptake in water mass during the initial stage of DNA elution for the stronger adsorbing conditions, which suggests that for these conditions the DNA film is partly dehydrated during the prior adsorption process. Overall, several positively charged and polar neutral amino acid buffers show promise as an alternative to methods based on chaotropic salts for solid phase DNA extraction.
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