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PubMed1993

Micropreparative two-dimensional electrophoresis allowing the separation of samples containing milligram amounts of proteins.

We describe some simple modifications to the micropreparative two-dimensional (2-D) electrophoresis procedure using immobilized pH gradients in the first dimension and sodium dodecyl sulfate-electrophoresis in the second dimension. The geometry of the immobilized pH gradient strips has been changed to allow the use of large sample application cups that can accommodate greater sample volumes. The use of narrow range immobilized pH gradients with a large sample loading volume allows an efficient resolubilization of polypeptides after the first dimension. As a result, the vertical streaking caused by too high a protein concentration is eliminated in the second dimension. Protein identification by N-terminal sequencing is facilitated by the large protein load (1-15 mg) which can be employed using this modification. Spots not normally detectable on conventional analytical 2-D maps, even with sensitive silver staining, are observed. Results for plasma and liver proteins are shown.

Blood Proteins↗
PubMed1994

Separation and sequencing of familiar and novel murine proteins using preparative two-dimensional gel electrophoresis.

Strategies are needed for rapid protein isolation in order to identify disease-related proteins and facilitate the design of oligonucleotides for further molecular inquiry. In our laboratory, C3H10T1/2 murine fibroblasts have been found to express a variety of proteins in various subcellular fractions which are relevant to experimental transformation and carcinogenesis. Preparative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) procedures were developed to identify major cytoplasmic proteins by electroblotting and microsequencing. Isoelectric focusing tube gels were enlarged to 6 mm ID to accommodate larger protein loads at 0.5 to 2 mg protein. Separated proteins were electrotransferred from 6 mm thick slab gels onto 0.22 mu polyvinylidene difluoride membranes. Nearly 100 prominent blotted proteins were stained with Coomassie Brilliant Blue between pI 4.5-7.0 and 18-106 kDa and, of these, 27 prominent and well-resolved proteins were selected for sequencing. Sequences of 14 to 24 amino acid residues in length were obtained from 11 proteins which were identified from computerized databases. Some of these identified proteins had structural or enzymatic functions while others had only recently been discovered, including a newly reported Hsp 70 class member and a novel calcium-binding protein, reticulocalbin. The new heat shock protein has a molecular mass of 75 kDa and has been designated as Grp75, PBP74, CSA or p66mot-1 in mice and humans with purported roles in transformation and antigen processing. Reticulocalbin is an endoplasmic reticular protein which contains six domains of the EF-hand motif associated with high-affinity calcium-binding proteins. It may be involved in protein transport and luminal protein processing. In addition, sequences of 5 to 11 residues in length were also obtained from six other unidentified proteins. Thus, we have found that preparative 2-D PAGE serves as a powerful one-step purification method for protein isolation and characterization from an important in vitro murine model for the study of carcinogenesis.

Amino Acid Sequence↗
PubMed1994

Method for rapid electrophoretic transfer of isoelectric focusing gels to polyvinylidene difluoride.

We present here a protocol for unidirectional wet electrotransfer of isoelectric focusing (IEF) gels to polyvinylidene difluoride (PVDF) that can be carried out in < 2 h, including preequilibration steps. In developing this protocol, we systematically evaluated sodium dodecyl sulfate and methanol concentrations in the preequilibration washes and length of electrotransfer. We demonstrated that the method is useful for tubulin, a protein that resists usual semi-dry transfer procedures due to its limited solubility under IEF conditions. We also successfully used our method for transferring the microtubule-associated protein, tau, as well as carbamylyte standards, creatine phosphokinase and carbonic anhydrase.

Acrylic Resins↗
PubMed1995

A preparative method for sequencing proteins and peptides: in situ gel staining with subsequent passive elution onto polyvinylidine difluoride membranes.

A preparative method for obtaining both N-terminal and internal peptide amino acid sequences from purified proteins is reported. The methodology reliably yields high fidelity signal from between 14 to 30 residues per purified protein or peptide, with low backgrounds on amino acid analysis. The procedure relies on the use of in situ staining of proteins during preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the utilisation of microconcentrators to repeatedly concentrate small amounts of proteins onto a small polyvinylidene difluoride (PVDF) disc until sufficient amounts have been adsorbed so as to give a strong sequencing signal. The protein elution and subsequent adsorption can be monitored visually with a dye and the final product, a PVDF disc with the adsorbed protein or peptide, can be directly inserted into the automated amino acid sequencer.

Amino Acid Sequence↗
PubMed1995

Sensitive quantification of proteins by electrophoresis in gels by use of chemiluminescence.

For protein quantitation in gels or blotting membranes, chemiluminescence (CL) offers the advantages of a substantial improvement of detection limits. Easy-to-use CL-chemicals and specific probes such as antibodies conjugated to enzymes, e.g. alkaline phosphatase (AP) may be used in combination with a newly developed luminometer. CL was found to have low detection limits and a linear relation between relative light units (RLU) and the concentration of the antibody enzyme complex present over a wide concentration range. Measurements of the immunoglobulin IgE in dot blots and in blots after sodium dodecyl sulfate (SDS) electrophoresis under nonreducing conditions in agarose gels are described.

Alkaline Phosphatase↗
PubMed1996

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and centrifuge blotting: preparation of polypeptides for amino-terminal sequence analysis.

The applicability and reproducibility of a previously described (L. F. Hermansen et al., Electrophoresis 1993, 14, 1302-1306) centrifuge-blotting procedure for capturing subnanomolar amounts of protein on polyvinylidene difluoride membranes for direct Edman degradation was further investigated. Proteins with different molecular masses were centrifuge-blotted onto Immobilon CD membranes. Simultaneous blotting and desalting was achieved with an overall yield of 15-56% after 2 h centrifugation for proteins with a molecular mass of 12-30 kDa. Centrifugation of myoglobin for 6 h resulted in an overall yield of 72%. The subnanomolar amounts obtained were also sufficient to conduct cyanogen bromide cleavage in situ on proteins with blocked NH2-terminus and to generate sequence information.

Centrifugation↗
PubMed1996

Identification of stress proteins in lysates of human cell lines separated by two-dimensional electrophoresis and electroblotted simultaneously onto two different membranes.

A protocol based on a combination of established methods for the characterization and identification of inducible stress proteins in human cell lines is described. A particular protein spot, collected from several micropreparative two-dimensional electrophoresis (2-DE) gels, is concentrated into a new gel prior to simultaneous electrotransfer onto a Cationic Durapore (CD) membrane and onto a polyvinylidene (PVDF) backup membrane. The protein blotted onto the PVDF support is subjected to N-terminal sequence analysis. From the protein bound to the CD membrane the peptide mass profile is obtained by proteolytic digestion of the protein followed by the separation of the resulting peptides by high performance liquid chromatography (HPLC) and their detection by on-line electrospray mass spectrometry (LC/MS). Additional internal sequence information may be obtained by amino acid sequence analysis of peptides collected in the HPLC effluent. The efficiency of this strategy is demonstrated with two proteins extracted from 15 micropreparative 2-DE gels of an extract of a human liver cell line. The peptide mass fingerprinting of a 60 kDa protein with a pI of 5.3 assigned 22 of 37 peptides to the heat shock protein 60 (Hsp 60). The result was confirmed by the N-terminal sequence analysis of the undigested protein and of an internal tryptic fragment. The second sample, a 40 kDa protein with a pI of 4.9, was identified as a processed form of the heat shock cognate 71 kDa protein (Hsc 70).

Amino Acid Sequence↗
PubMed1996

Ultraviolet matrix assisted laser desorption ionization-mass spectrometry of electroblotted proteins.

Direct mass spectrometric analysis of proteins electroblotted onto polyvinylidene fluoride membranes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis is demonstrated by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) with a linear time-of-flight instrument, equipped with a nitrogen laser (337 nm). The blotted proteins were desorbed directly from the blotting membrane after incubation with sinapinic acid as matrix. Different commercially available membranes resulted in high quality protein signals for hydrophobic membranes exhibiting high specific surface areas (Immobilon PSQ or Trans-Blot) or for charged membranes (Immobilon CD). Systematic investigations with standard proteins were performed to compare standard preparation procedures for ultraviolet (UV) MALDI-MS on stainless steel sample stages and preparation of proteins immobilized onto membranes either by direct application from protein solutions (spotting) or by electrotransfer from gels (electroblotting). Aspects such as mass resolution reproducibility from shot to shot and spot to spot, mass accuracy, and preservation of protein localization are addressed in this paper.

Electrophoresis, Polyacrylamide Gel↗
PubMedDate not supplied

Comparison of in-gel and on-membrane digestion methods at low to sub-pmol level for subsequent peptide and fragment-ion mass analysis using matrix-assisted laser-desorption/ionization mass spectrometry.

The success of the mass spectrometric-based approaches for the identification of gel-separated proteins relies upon recovery of peptides, without high levels of ionization-suppressing contaminants, in solvents compatible with the mass spectrometer being employed. We sought to determine whether in-gel or on-membrane digestion provided a significant advantage when low to sub-pmol quantities of gel-separated proteins were analyzed by matrix-assisted laser-desorption/ionization mass spectrometry (MALDI-MS) with respect to the number and size of released peptides. Serial dilutions of five standard proteins of M(r) 17,000 to 97,000 (from 16 pmol to 125 fmol) were electrophoresed and subjected to in-gel digestion (using a microcolumn clean-up protocol, Courchesne, P.L. and Patterson, S. D., BioTechniques, 1997, in press) or on-membrane digestion following blotting to the PVDF-based membranes, Immobilon-P and Immobilon-CD. Peptide maps were able to be obtained for all proteins at the detection limit of each method (Immobilon-P and Immobilon-CD, 0.5 pmol; and in-gel, 125 fmol), and searches of Swiss-Prot or a non-redundant database (> 193000 entries) successfully identified all of the proteins, except beta-casein. Fragment-ion spectra using a curved-field reflector MALDI-MS were obtained from more than one peptide per protein at loads down to 250 fmol (except beta-casein). Using the uninterpreted data, a search of the nonredundant database and a six-way translation of GenBank dbEST (> 2,208,000 entries total) was able to identify myoglobin, carbonic anhydrase II, and phosphorylase b.

Acrylic Resins↗
PubMedDate not supplied

Proteome analysis of glycoforms: a review of strategies for the microcharacterisation of glycoproteins separated by two-dimensional polyacrylamide gel electrophoresis.

Preparative two-dimensional polyacrylamide electrophoresis (2-D PAGE) is a method of separation which for the first time allows protein isoforms to be readily purified for subsequent analysis. The profile of the 2-D separation of the protein complement (proteome) of eukaryotic cells and tissues typically contains obvious 'trains' of spots which differ in pI and/or apparent molecular mass. These are usually isoforms of the same protein and result from post-translational modifications. There is growing evidence that alterations to the glycosylation and/or phosphorylation of a protein can be correlated with developmental and pathological changes; these changes can be visualised on the 2-D separation. It is not clear, however, how these modifications alter the structural properties of the protein and affect their migration in this mode of separation. Strategies need to be developed to obtain a more detailed understanding of the reason for the appearance of isoforms as discrete spots on 2-D PAGE. Standard proteins, fetuin and ovalbumin, were used to monitor the effect of the removal of glycans and phosphates on the migration of the glycoproteins in the 2-D system. The isoforms were not simply explained by the presence or absence of a single modification. To further investigate the reasons for the different migration of the isoforms it is necessary to characterise the modifications in more detail. Unlike protein analysis, until recently the available methodology for the analysis of the glycans attached to proteins has not been sensitive enough to allow analysis of single spots in gels or blots resulting from 2-D electrophoresis. In this paper we review current and future strategies for characterisation of protein modifications using single spots from 2-D gels.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1997

Combined immunostaining and Coomassie Brilliant Blue staining of polyvinylidene difluoride membranes without organic solvent.

A method for staining proteins on polyvinylidene difluoride membranes without using organic solvent is described. The method uses preblocking of the membrane with either Tween 20 or polyethylene glycol followed by staining with 0.01% Coomassie Brilliant Blue. No destaining of the membrane is needed afterwards. Preblocking with polyethylene glycol is compatible with microsequencing while Tween 20 leads to very low initial yields. Preblocking with Tween 20 has the additional advantage of allowing immunostaining followed by Coomassie Brilliant Blue staining for total protein on the same membrane.

Electrophoresis, Polyacrylamide Gel↗
PubMed1997

A new method to assign immunodetected spots in the complex two-dimensional electrophoresis pattern.

A new, easy method for the immunodetection of specific antigens in two-dimensional electrophoresis (2-DE) is described. Areas of 2-DE gels containing antigens of interest are electrophoretically transferred to polyvinylidene difluoride membranes, immunostained with specific antibodies using Fast Red or 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium as detection systems and counterstained with Coomassie Brilliant Blue. In contrast to conventional methods, it is possible to use this procedure to exactly assign immunoreacting proteins on a single blot to their corresponding and surrounding blue-stained protein spots.

Animals↗
PubMed1997

Elution concentration of proteins cut from two-dimensional polyacrylamide gels using Pasteur pipettes.

The present study devised a new procedure for concentrating proteins cut from primary two-dimensional polyacrylamide gels prior to their structural characterization. Gel spots containing a protein were cut from several identical two-dimensional gels and loaded on the top of a high tensile strength stacking gel in a Pasteur pipette. The protein was concentrated electrophoretically into a small volume in the narrow tip of the pipette. The concentrated protein was then blotted from the stacking gel onto a polyvinylidene difluoride membrane, where the protein was subjected to tryptic on-membrane digestion. Utilizing this system, we identified 22 proteins chosen randomly from two-dimensional gels of proteins from rat dermal papilla cells by internal microsequencing. As much as 1.5 mL volume (cut gel spots in protein sample buffer) could be loaded onto the Pasteur pipettes, generally yielding a final on-membrane area of approximately 2 mm2 after elution concentration and electroblotting onto polyvinylidene difluoride membranes. We concluded that this newly devised system is effective and useful for concentrating proteins prior to structural characterization, and that larger volumes than previously recommended can be effectively concentrated, with little or no effect on the final on-membrane area occupied by the concentrated proteins.

Amino Acid Sequence↗
PubMed1998

Elution of glycoproteins from replicas of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels.

A method for the elution of glycoproteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) replicas of gels on polyvinylidene difluoride (PVDF) membranes is described. Ten model glycoproteins were resolved by SDS-PAGE and then electrotransferred onto PVDF membranes. After reversible staining, glycoprotein bands were eluted with a mixture of SDS/Triton X-100 at pH 9 or with a mixture of guanidinium hydrochloride/lysophosphatidylcholine at neutral pH. For both types of eluents, the final recoveries ranged from over 30% to about 80%. Good recoveries and mild conditions of elution render the method applicable for the structural elucidation of glycan chains.

Acrylic Resins↗
PubMed1998

Preparative isoelectric focusing and preparative electrophoresis of hydrophobic Candida albicans cell wall proteins with in-line transfer to polyvinylidene difluoride membranes for sequencing.

Hydrophobic proteins in the cell wall of the opportunistic fungal pathogen Candida albicans are involved in adhesion of this organism to host tissue and thus play a role in its pathogenicity. The hydrophobic nature of these proteins results in their loss during purification due to adsorption to apparatus surfaces. This problem, combined with their low abundance, has made it problematic to purify the hydrophobic proteins in sufficient quantity for sequencing or biochemical analysis. We describe a system that combines preparative isoelectric focusing with continuous elution preparative electrophoresis. The system provides a two-dimensional protein separation while maintaining protein solubility and minimizing protein loss due to adsorption. In addition, we have added an in-line transfer of electrophoretic fractions directly to polyvinylidene difluoride (PVDF) membranes, which further reduces both exposure to apparatus surfaces and purification time.

Blotting, Western↗
PubMed1998

Analyzing glycoproteins separated by two-dimensional gel electrophoresis.

Two-dimensional (2-D) electrophoresis is the preferred method for separating the glycoforms of proteins. The isoforms usually present as 'trains' of spots in the first dimension and may also differ in molecular weight. The primary goal for analyzing the carbohydrate content of glycoprotein spots is to understand the 'rules' which govern the migration of glycoproteins in 2-D electrophoresis. These rules can then be used to produce predictive vectors to interpret changes in glycosylation patterns. Techniques for the analysis of oligosaccharides released from glycoproteins which have been electroblotted to PVDF membrane after one-dimensional (1-D) and 2-D preparative gel electrophoresis are described. The oligosaccharides are removed enzymatically (PNGase F of N-linked oligosaccharides) or chemically (beta-elimination of O-linked oligosaccharides) and separated by high performance anion exchange chromatography (HPAEC-PAD) and identified by electrospray ionization mass spectrometry (ESI-MS) or analyzed directly by ESI-MS. After enzymic removal of the N-linked oligosaccharides the protein spots can be further analyzed by Edman sequence tagging for identification and quantitation of the protein and by acid hydrolysis for monosaccharide analysis of the O-linked oligosaccharides. These approaches have been proved on 1-D PAGE electroblotted bovine fetuin and human glycophorin A and then used to analyze two abundant proteins which separate as glycoforms on 2-D PAGE preparative narrow range (pH 4.5-5.5) blots of human plasma: alpha2-HS glycoprotein (human fetuin) and alpha1-antitrypsin (alpha1-protease inhibitor). It is apparent that both the macroheterogeneity (site occupation) and microheterogeneity (diversity of structures) of the glycosylation contribute to the separation of protein isoforms in 2-D PAGE.

Amidohydrolases↗
PubMed1998

Routine identification of proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels or polyvinyl difluoride membranes using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS).

As the resource laboratory for Rockefeller University our emphasis continues to be on methodology development for the routine analysis of low abundance proteins isolated from native sources. In the past ten years, gel electrophoresis of proteins has become the method of choice for the preparation of microgram and submicrogram quantities of protein for primary structural characterization, and over 95% of the samples submitted for protein identification are either in a gel or bound to polyvinyl difluoride membranes (PVDF). As such, we employ multiple microanalytical approaches encompassing Edman sequence degradation, amino acid and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometric analysis to provide an integrated protein characterization of such samples. Here we describe the two major services we employ when providing protein identification from in-gel or PVDF-bound proteins.

Amino Acid Sequence↗
PubMed1998

'98 Escherichia coli SWISS-2DPAGE database update.

The combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), computer image analysis and several protein identification techniques allowed the Escherichia coli SWISS-2DPAGE database to be established. This is part of the ExPASy molecular biology server accessible through the WWW at the URL address http://www.expasy.ch/ch2d/ch2d-top.html . Here we report recent progress in the development of the E. coli SWISS-2DPAGE database. Proteins were separated with immobilized pH gradients in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. To increase the resolution of the separation and thus the number of identified proteins, a variety of wide and narrow range immobilized pH gradients were used in the first dimension. Micropreparative gels were electroblotted onto polyvinylidene difluoride membranes and spots were visualized by amido black staining. Protein identification techniques such as amino acid composition analysis, gel comparison and microsequencing were used, as well as a recently described Edman "sequence tag" approach. Some of the above identification techniques were coupled with database searching tools. Currently 231 polypeptides are identified on the E. coli SWISS-2DPAGE map: 64 have been identified by N-terminal microsequencing, 39 by amino acid composition, and 82 by sequence tag. Of 153 proteins putatively identified by gel comparison, 65 have been confirmed. Many proteins have been identified using more than one technique. Faster progress in the E. coli proteome project will now be possible with advances in biochemical methodology and with the completion of the entire E. coli genome.

Bacterial Proteins↗