Simplified techniques for elution of proteins from polyacrylamide gel, staining, destaining, and isoelectric focusing

A simple and rapid electrophoretic method for the simultaneous elution of proteins in high yields from polyacrylamide gel slabs is described. The apparatus is simple and easily constructed. The method involves vertical elution from a horizontally placed gel across its thickness into buffer-soaked polyurethane foam. Even the slow-moving, high-molecular-weight protein ferritin is eluted in 1 h. The technique can also be used for rapid destaining as well as for simultaneous staining and destaining of the polyacrylamide gel which are completed in 15 and 30 min, respectively. Polyurethane foam strips have also been used to simplify the preparative isoelectric focusing technique and the subsequent elution of protein bands, obviating the need for Ultrodex, fractionating grid, sample applicator, and elution columns.     

A rapid and effecient method for the isolation of proteins from polyacrylamide cells.

A procedure is described for the rapid and efficient electrophoretic elution of protein from polyacrylamide gels which is then collected in a dialysis bag tied to the end of a tube containing the gel slices. To illustrate the method a heterogeneous preparation of alkaline phosphatase was used from which a single homogeneous component was isolated in six hours with a recovery of 86%. The eluted protein is collected in a volume which can easily be kept below 1.5 ml, thus eliminating the need for subsequent concentration. The method has also been used successfully in two other systems in which a human lung tumor-associated antigen and glycogen synthetase from yeast were isolated. Since the method utilizes a standard analytical gel electrophoresis apparatus with no modifications or accessories, it should be immediately applicable for the isolation of many different proteins from polyacrylamide gels.     

A Rapid and Efficient Method for the Isolation of Proteins from Polyacrylamide Gels

A procedure is described for the rapid and efficient electrophoretic elution of protein from polyacrylamide gels which is then collected in a dialysis bag tied to the end of a tube containing the gel slices. To illustrate the method a heterogeneous preparation of alkaline phosphatase was used from which a single homogeneous component was isolated in six hours with a recovery of 86%. The eluted protein is collected in a volume which can easily be kept below 1.5 ml, thus eliminating the need for subsequent concentration. The method has also been used successfully in two other systems in which a human lung tumor-associated antigen and glycogen synthetase from yeast were isolated. Since the method utilizes a standard analytical gel electrophoresis apparatus with no modifications or accessories, it should be immediately applicable for the isolation of many different proteins from polyacrylamide gels.     

Mapping of radiolabeled peptides derived from proteolysis of polypeptides bound to nitrocellulose after "Western" blotting

Sections of nitrocellulose containing bound 32P-labeled polypeptides were excised from "Western" blots and exhaustively digested by trypsin in order to analyze the distribution of phosphorylation sites between the products of limited proteolysis of the multifunctional protein CAD. Using the criterion of analytical isoelectric focusing, the 32P-peptides obtained by this method were found to be similar, although not identical, to peptides obtained by a more conventional digestion of trichloroacetic acid precipitates. Digestion on Western blots is more straightforward than electrophoretic elution of individual gel slices, gives better recoveries than direct digestion of gel slices, and is particularly suitable for peptide mapping of small peptides which bind to nitrocellulose but would diffuse out of polyacrylamide gels during the commonly used fixing and staining procedures.     

Mass spectrometric quantitation of peptides and proteins using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA)

A method (denoted SISCAPA) for quantitation of peptides in complex digests is described. In the method, anti-peptide antibodies immobilized on 100 nanoliter nanoaffinity columns are used to enrich specific peptides along with spiked stable-isotope-labeled internal standards of the same sequence. Upon elution from the anti-peptide antibody supports, electrospray mass spectrometry is used to quantitate the peptides (natural and labeled). In a series of pilot experiments, tryptic test peptides were chosen for four proteins of human plasma (hemopexin, alpha1 antichymotrypsin, interleukin-6, and tumor necrosis factor-alpha) from a pool of 10,203 in silico tryptic peptide candidates representing 237 known plasma components. Rabbit polyclonal antibodies raised against the chosen peptide sequences were affinity purified and covalently immobilized on POROS supports. Binding and elution from these supports was shown to provide an average 120-fold enrichment of the antigen peptide relative to others, as measured by selected ion monitoring (SIM) or selected reaction monitoring (SRM) electrospray mass spectrometry. The columns could be recycled with little loss in binding capacity, and generated peptide ion current measurements with cycle-to-cycle coefficients of variation near 5%. Anti-peptide antibody enrichment will contribute to increased sensitivity of MS-based assays, particularly for lower abundance proteins in plasma, and may ultimately allow substitution of a rapid bind/elute process for the time-consuming reverse phase separation now used as a prelude to online MS peptide assays. The method appears suitable for rapid generation of assays for defined proteins, and should find application in the validation of diagnostic protein panels in large sample sets.     

Recovery of polypeptides from polyacrylamide gels by electrophoretic elution in a centrifugation concentrator

A method for recovery of polypeptides from polyacrylamide gels by electrophoretic elution in a commercially available concentrator, the Amicon-Centricon sample reservoir, has been devised. The recoveries were greater than 90% with four different polypeptides tested (12.5 to 80 kDa). After elution, sample concentration or salt exchange can be carried out without sample transfer. There were no loss of sample during the postelution procedures when the elution buffer was replaced by 0.01 or 0.05% sodium dodecyl sulfate.     

Electroelution of antigens immobilized on antibody-linked affinity matrices

An electrophoretic elution procedure for desorption of antigens from antibody-linked gel matrices is described which is performed in a commercially available elution device. The technique presented does not involve denaturing conditions such as chaotropic reagents or low and high pH elution buffers. Comparison of electroelution and conventional elution with acidic buffer reveals that the techniques is especially suitable for monoclonal antibodies with high affinity for their ligands. Recovery of antigens after electroelution is significantly higher than by desorption with glycine-HCl, pH 2.5. There is no loss of antigens during post-elution procedures. Proteins are obtained in small elution volumes and can be desalted without sample transfer.     

Rapid isolation of high-molecular-weight DNA from agarose gels

We have developed a simple, reliable, and rapid method for recovering DNA from agarose gels. While many methods for DNA extraction have already been described, few provide quantitative recovery of large DNA molecules. These procedures generally require costly apparatus, extended elution times, or considerable handling of the sample after elution. Our method employs a novel electroelution chamber constructed from acrylic plastic. Gel slices containing DNA are placed in the chamber between platinum electrodes. Voltage is applied and a continuous flow of buffer sweeps the eluted DNA from the chamber into an external receptacle. Elution is complete in 7 min. Concentrated DNA is obtained by butanol extraction and alcohol precipitation in 1 h. Recoveries, quantitated by counting radiolabeled DNA or by densitometry of analytical gels, were 94 to 100% for fragments of 4 to 50 kb. The eluted DNA was undegraded and could be digested with restriction enzymes, ligated, end-labeled, or used to transform cells as efficiently as noneluted DNA. Complete elution of a 100-kb plasmid, a 194-kb concatemer of bacteriophage lambda, and of 440- and 550- chromosomes of Saccharomyces cerevisiae was also achieved using the same process. This method is suitable for routine use in a wide range of cloning applications, including the electrophoretic isolation of large DNA molecules.     

Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a positively charged membrane filter

Zeta-bind, a positively charged nylon membrane, was tested as an immobilizing matrix for the electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels. It was found that Zeta-bind has a considerably greater capacity than does nitrocellulose for protein binding. Because of this property, more efficient elution of proteins from gels can be used (by omitting methanol from transfer buffers). The procedure described is more amenable to quantitation than usual nitrocellulose-based transfer. Antibody or lectin overlay techniques are also more sensitive on Zeta-bind than on nitrocellulose.     

Electrofractionation: a technique for detecting and recovering biomolecules.

Electrofractionation (EF) is a technique that allows electrophoretic materials to be detected and recovered following electrophoresis. The EF apparatus utilizes the resolving power of electrophoresis and the mobile phase of liquid chromatography to create a continuous elution system. Our data indicate that EF is able to detect and recover oligonucleotides, as DNA fragments, proteins, and presumably other material that can be analyzed by electrophoresis. EF shares functional similarities with high-performance electrophoresis chromatography (HPEC) but operates by a different strategy and at a fraction of the cost. Moreover, EF can be constructed largely from standard laboratory equipment. The simplicity, rapid analysis times, low cost, and high recovery yields of EF make this system a practical alternative to the conventional detection and purification methods used for biomolecules.     

Advanced analytical methods for hemoglobin variants

Hemoglobin variants are the protein mutations most often encountered in the clinical scene. They have been useful for developing methods to analyze mutant proteins because of their size and ease of collection in large amounts. Improvements in analytical methods have been directed toward higher resolution in electrophoresis and shorter elution times in chromatography. More importantly, in the last 20 years, hemoglobin variants have been used in the development of mass spectrometric strategies for analyzing protein mutations. This approach consists of a series of steps: measurement of the molecular mass of globins to detect or confirm the presence of mutations, peptide mass mapping or peptide mass fingerprinting of an enzymatic digest to identify mutated peptides, and tandem mass spectrometry to determine or confirm the site and type of mutation. The mass spectrometric strategy has enabled rapid analysis and demonstrated a superb ability to detect a number of hemoglobin variants, particularly those without a change in electrophoretic or chromatographic properties. Even with the recent advances in DNA analysis, protein analysis is still essential, because post-translational modifications following amino acid substitutions can occur including N-terminal acetylation, deamidation and oxidation-mediated processes.     

One-Step Purification of Recombinant Proteins Using a Nanomolar-Affinity Streptavidin-Binding Peptide, the SBP-Tag

We describe the use of the SBP-tag, a new streptavidin-binding peptide, for both the one-step purification and the detection of recombinant proteins. The SBP-tag sequence is 38 amino acids long and binds to streptavidin with an equilibrium dissociation constant of 2.5 nM. We demonstrate that a single-step purification of SBP-tagged proteins from bacterial extract yields samples that are more pure than those purified using maltose-binding protein or the His-tag. The capacity of the immobilized streptavidin used to purify SBP-tagged proteins is about 0.5 mg per milliliter of matrix, which is high enough to isolate large quantities of proteins for further study. Also, the elution conditions from the streptavidin column are very mild and specific, consisting of the wash buffer plus biotin. This combination of high-affinity, high-yield, mild elution conditions, and simplicity of use makes the SBP-tag suitable for high-throughput protein expression/purification procedures, including robotically manipulated protocols with microtiter plates. Additionally, the SBP-tag can be used for detection since a wide variety of streptavidin-conjugated fluorescent and enzymatic systems are commercially available. We also present a new, rapid, method for the measurement of protein-protein, protein-peptide, or protein-small molecule equilibrium dissociation constants that require as little as 1 fmol of labeled protein. We call this method the spin-filter binding inhibition assay.     

A simple and fast electrophoretic method for elution of nucleic acids from gels

A very convenient electrophoretic procedure for DNA or RNA elution from agarose or polyacrylamide gels is described. The gel piece with nucleic acid to be eluted is contained in a dialysis bag filled with buffer and elution is carried out in a horizontal electrophoresis apparatus. The nucleic acid is recovered with a high yield and can be used, without prior treatment, in further enzymatic or chemical reactions. Results obtained with DNA are presented here.     

Electrophoretic affinity chromatography: method validation

A new method for preparative-scale separation of biomolecules, electrophoretic affinity chromatography (EAC), is proposed in this paper. Separation by EAC is carried out in a long and ribbon-like multicompartment electrolyser separated by membranes, in which the two central compartments are used for packing the gel matrix and for sample loading respectively. Next to the central compartments are the elution compartments and electrode compartments. The electric field is applied perpendicular to the fluid flow in the compartments. Adsorption and desorption steps may both be carried out in the presence of an electric field, which transports the target components into the gel compartment for adsorption and the impurities into the elution compartments for washing. After the adsorption step an elution solution is introduced and the product is released from the gel matrix and washed out. Separation of human serum albumin (HSA) from human serum gives HSA product of high purity, as demonstrated by isoelectric focusing analysis. The characteristics of electrophoretic binding of HSA on Blue Sepharose Fast Flow are examined. The preliminary results show that this new method has advantages in terms of high rate of mass transfer and ease of scaling up, which are of particular interest when large-scale separation of biomolecules is considered.     

Anionic glutathione S-transferases of human erythrocytes, placenta, and lung: evidence for structural differences

Studies were undertaken to elucidate the structural interrelationships among glutathione S-transferase (GST) isozymes of human placenta, lung, and erythrocytes. Results of the high-performance liquid chromatography of the trypsin digests of the three isozymes indicate minor but significant differences in their elution profiles. Although a number of peptides generated by proteolysis were common for either 2 or 3 of the isozymes, significant differences were observed in elution profiles of other peptides. Qualitative as well as quantitative differences were also observed in the electrophoretic peptide maps of these isozymes. These studies suggest that there may be fine structural differences among the pi class GST isozymes of human tissues.     

SP-Sephadex equilibrium chromatography of bradykinin and related peptides: Application to trypsin-treated human plasma

An analytical method is deseribed for the separation of bradykinin, Lys-bradykinin, and Met-Lys-bradykinin by equilibrium chromatography on SP-Sephadex C-25 eluted in 0.02 m Tris-HCl buffer, pH 8.10, 0.12 m NaCl. A second elution buffer, 0.02 m Tris-HCl buffer, pH 7.70, 0.06 m NaCl, serves as a second parameter for the identification of bradykinin and also separates the hormone from plasma bradykinin-potentiating peptides. Ten to one-hundred nanomoles of each peptide can be recovered in high yields, identified by elution position, and measured by bioassay with the isolated guinea pig ileum. The identification of bradykinin as the peptide released by trypsin acting on acid-denatured plasma is documented as an illustration of the method.     

Iodine stain for detection of peptides after isoelectric focusing

Iodine stain is used for the detection of peptides after isoelectric focusing has been developed. Ultrathin gels (240–360 μm) are cast and, after focusing, dried at 110°C on a filter paper sheet. The paper-pasted gel is then exposed to iodine vapors for a few seconds to a few minutes, depending on the peptide load. White peptide zones are visivle on a brown, uniform background. The reaction is fully reversible and can be used also for small-scale preparative purification of peptides. Better than 80% recoveries of peptide from the gel can be obtained by elution in 80% acetic acid.     

Application of peptide LC retention time information in a discriminant function for peptide identification by tandem mass spectrometry

We describe the application of a peptide retention time reversed phase liquid chromatography (RPLC) prediction model previously reported (Petritis et al. Anal. Chem. 2003, 75, 1039) for improved peptide identification. The model uses peptide sequence information to generate a theoretical (predicted) elution time that can be compared with the observed elution time. Using data from a set of known proteins, the retention time parameter was incorporated into a discriminant function for use with tandem mass spectrometry (MS/MS) data analyzed with the peptide/protein identification program SEQUEST. For singly charged ions, the number of confident identifications increased by 12% when the elution time metric is included compared to when mass spectral data is the sole source of information in the context of a Drosophila melanogaster database. A 3-4% improvement was obtained for doubly and triply charged ions for the same biological system. Application to the larger Rattus norvegicus (rat) and human proteome databases resulted in an 8-9% overall increase in the number of confident identifications, when both the discriminant function and elution time are used. The effect of adding "runner-up" hits (peptide matches that are not the highest scoring for a spectra) from SEQUEST is also explored, and we find that the number of confident identifications is further increased by 1% when these hits are also considered. Finally, application of the discriminant functions derived in this work with approximately 2.2 million spectra from over three hundred LC-MS/MS analyses of peptides from human plasma protein resulted in a 16% increase in confident peptide identifications (9022 vs 7779) using elution time information. Further improvements from the use of elution time information can be expected as both the experimental control of elution time reproducibility and the predictive capability are improved.     

A method for the rapid and efficient elution of native affinity-purified protein A tagged complexes

A problem faced in proteomics studies is the recovery of tagged protein complexes in their native and active form. Here we describe a peptide, Bio-Ox, that mimics the immunoglobulin G (IgG) binding interface of Staphylococcus aureus Protein A, and competitively displaces affinity-purified Protein A fusion proteins and protein complexes from IgG-Sepharose. We show that Bio-Ox elution is a robust method for the efficient and rapid recovery of native tagged proteins, and can be applied to a variety of structural genomics and proteomics studies.     

Electrophoretic elution of nucleic acids from acrylamide and agarose gels

A simple method for electrophoretic elution of nucleic acids from gel slices is described. The procedure utilizes a standard tube gel system and can be completed in as little as one hour. Nucleic acids are recovered in a small volume with almost 100% efficiency. The procedure is applicable equally to acrylamide and agarose gels, and small as well as large RNA and DNA molecules. The eluted nucleic acids are essentially undegraded and are suitable for a variety of structural and biological analyses.