A simplified method of protein electroblotting after isoelectric focusing on an ultrathin polyacrylamide gel layer fixed on a cellophane support.

A method is described for isoelectric focusing of proteins, using an ultrathin-layer polyacrylamide gel on cellophane, followed by electrophoretic transfer of separated proteins onto a nitrocellulose membrane. The polyacrylamide gel is firmly attached to the cellophane and thus protected from mechanical damage; such gels are easily manipulated. Cellophane is permeable to ions and application of this gel support overcomes difficulties resulting from the removal of ultrathin gels from a plastic support on electroblotting. Proteins separated under nondenaturing conditions were transferred onto a nitrocellulose membrane and detected by the concanavalin A-peroxidase technique. The proposed approach makes it possible to analyze the variability of nondenatured proteins and glycoproteins of different origin.     

Direct determination of selenoproteins in polyvinylidene difluoride membranes by electrothermal atomic absorption spectrometry

A method for the direct determination of selenoproteins in plastic membranes after protein separation by gel electrophoresis was developed. Quantification was based on the determination of the selenium content of the proteins by electrothermal atomic absorption spectrometry (ET-AAS) after manual introduction of membrane pieces into the graphite furnace. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane by semi-dry electroblotting. After staining the membrane, the protein bands were excised and chemical modifier was added on top of the excised membrane prior to atomic absorption measurement. Acceptable linearity was achieved in the range 2-10 ng Se, corresponding to selenium concentrations close to 1 mg/L, when aqueous solutions of selenomethionine standard as well as selenoprotein standard were applied to the membrane. A characteristic mass of 54 +/- 4 pg/0.0044 s was obtained for the selenoprotein standard. Protein transfer from polyacrylamide gel to the membrane was quantitative and no interferences were introduced. The method was used for identification of selenoprotein P after enrichment of the protein from human plasma.     

Direct determination of selenoproteins in polyvinylidene difluoride membranes by electrothermal atomic absorption spectrometry

A method for the direct determination of selenoproteins in plastic membranes after protein separation by gel electrophoresis was developed. Quantification was based on the determination of the selenium content of the proteins by electrothermal atomic absorption spectrometry (ET-AAS) after manual introduction of membrane pieces into the graphite furnace. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane by semi-dry electroblotting. After staining the membrane, the protein bands were excised and chemical modifier was added on top of the excised membrane prior to atomic absorption measurement. Acceptable linearity was achieved in the range 2– 10 ng Se, corresponding to selenium concentrations close to 1 mg/L, when aqueous solutions of selenomethionine standard as well as selenoprotein standard were applied to the membrane. A characteristic mass of 54 ± 4 pg/0.0044 s was obtained for the selenoprotein standard. Protein transfer from polyacrylamide gel to the membrane was quantitative and no interferences were introduced. The method was used for identification of selenoprotein P after enrichment of the protein from human plasma. Received: 28 June 1999 / Revised: 14 September 1999 / Accepted: 16 September 1999     

High yield electroblotting onto polyvinylidene difluoride membranes from polyacrylamide gels.

Optimal conditions of electroblotting that led to high protein recovery on polyvinylidene difluoride (PVDF) membranes were determined for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS concentrations in the gel and transfer buffer were found to be the most important factors affecting the amount of protein recovered on the PVDF membrane. The largest loss occurred during the first 10-30 min of transfer due to the relatively high initial SDS concentration in the gel. During this initial stage of transfer, most of the protein passed through the primary membrane and was partially retained on secondary and tertiary membranes. The value of presoaking gels prior to transfer to reduce the amount of SDS was evaluated by quantitating free SDS densitometrically and by correlating the reduced SDS concentration with increased electroblotting efficiency from presoaked gels. Transfer time was evaluated and no "overtransfer" was found even after very long transfer times. These results clearly indicate that proteins electroblotted onto PVDF membranes were tightly bound and could not be released by extending the transfer time. The effects of methanol and SDS concentrations on protein adsorption from solution to PVDF were also determined quantitatively. The results of this study strongly suggest that proteins fully saturated with SDS cannot bind efficiently to PVDF membranes. Since SDS is necessary for high protein mobility, the challenge in efficient electroblotting is to maintain an optimal SDS concentration which is high enough to permit effective removal from the gel and low enough to permit effective binding to the PVDF membrane. For 1.5 mm thick gels containing 0.2% SDS, presoaking the gel for 15-20 min in transfer buffer with 10% methanol prior to electroblotting provided the best recovery on the primary membrane.     

Isolation and recovery of acidic oligosaccharides from polyacrylamide gels by semi-dry electrotransfer.

Acidic oligosaccharides derived from glycosaminoglycan heparin were separated by polyacrylamide gradient gel electrophoresis (PAGE). The gel could be visualized using Alcian Blue dye to give a pattern of highly resolved, well defined bands. The particular banding pattern obtained was the result of a heparinase catalyzed depolymerization which afforded oligosaccharide products that differed in size by one disaccharide unit. The separated oligosaccharides could be recovered prior to staining by electroelution onto a positively charged nylon membrane by a semi-dry transfer procedure. Subsequent elution and quantitative recovery of individual oligosaccharides from the membrane was achieved. By using multiple membrane layers a second separation dimension was obtained, resulting in increased oligosaccharide purity proportional to transfer depth. Preparative gradient polyacrylamide gel electrophoresis followed by semi-dry electro-transfer and recovery represents a novel method for the preparation of homogeneous acidic oligosaccharides.     

IMMUNOBLOT ANALYSIS OF CROSS-REACTIVITY OF MONOCLONAL ANTIBODIES AGAINST OAT PHYTOCHROME WITH PHYTOCHROME FROM SEVERAL PLANT SPECIES*

Abstract— Eighteen monoclonal antibodies (mAbs) have been produced against partially degraded phytochrome from A vena sativa cv. Trafalgar. Several of the mAbs are capable of recognising the antigen on immunoblots following sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and protein electroblotting. Six of these mAbs were screened for the ability to recognise electroblotted phytochrome from eight plant species. Particularly amongst the monocot species tested, but also amongst the dicots, the mAbs showed extensive cross-reactivity. The results suggest that there are perhaps several conserved antigenic sites on the phytochrome molecule.     

Microsequencing of proteins electrotransferred onto immobilizing matrices from polyacrylamide gel electrophoresis: application to an insoluble protein

Semidry electroblotting is convenient and allows a rapid and efficient protein transfer from one- or two-dimensional polyacrylamide gels onto sequencer stable supports for protein microsequence analysis in a gas-phase sequencer. Using this technique, we determined the amino acid sequences of the basic 7S globulin (Bg), an insoluble protein present in soybean seeds. Based on sequence determination, the cDNA-encoding Bg could be easily cloned and characterized.     

Fluorescence detection and quantitation of recombinant proteins containing oligohistidine tag sequences directly in sodium dodecyl sulfate-polyacrylamide gels

Two fluorophore-nitrilotriacetic acid conjugates, Pro-Q Sapphire 365 and Pro-Q Sapphire 488 oligohistidine gel stains, have been developed for the fluorescence detection of fusion proteins containing oligohistidine tags directly in sodium dodecyl sulfate polyacrylamide gels, without the requirement for electroblotting, reporter enzymes or secondary detection reagents. Pro-Q Sapphire 365 oligohistidine gel stain exhibits bright-blue fluorescence (emission maximum = 450 nm) when illuminated with UV-A or UV-B light from a standard ultraviolet transilluminator. Pro-Q Sapphire 488 oligohistidine gel stain exhibits bright-green fluorescence (emission maximum = 515 nm) when illuminated with visible light from a laser-based gel scanner equipped with a 470 nm second-harmonic generation (SHG) or 488 nm argon-ion laser source. Typically, 25-65 ng of oligohistidine-tagged fusion protein in whole cell lysates is detectable using either stain. After documenting the fluorescence signal from the Pro-Q Sapphire dyes, gels may be post-stained with the red-fluorescent SYPRO Ruby protein gel stain in order to reveal the total protein pattern.     

Direct N-terminal sequence analysis of rat liver plasma membrane glycoproteins separated by two-dimensional polyacrylamide gel electrophoresis

Nine previously uncharacterized membrane glycoproteins from normal rat liver have been analyzed by amino acid sequencing from two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) after transblotting to Immobilon-P membranes. Three of these components show altered levels of expression in liver tumors. A single electroblotted polyacrylamide gel yielded sufficient quantities of these glycoproteins for amino acid sequencing and the N-terminal structure could be determined for four of them. The remaining five glycoproteins of interest were not sequenceable in this manner, presumably because they had blocked N-termini. Prior to electrophoresis, two enrichment methods were applied to the crude liver membrane preparations: affinity chromatography with concanavalin A to isolate the plasma membrane glycoproteins and then fast protein liquid chromatography on Superose 12 to obtain components having a specific range of molecular weights. These materials were next subjected to 2-D PAGE using pH 4-6 carrier ampholytes in the first dimension and 7.5% sodium dodecyl sulfate gels in the second. The proteins were then electroblotted to Immobilon-P membranes and located by staining with Coomassie Brilliant Blue R-250. Our results demonstrate that N-terminal sequencing (gas-phase) can be achieved on polypeptides obtained from approximately 250 micrograms of total glycoproteins applied to a single 2-D gel.     

Development of a database of amino acid sequences for human colon carcinoma proteins separated by two-dimensional polyacrylamide gel electrophoresis

The tandem use of preparative two-dimensional polyacrylamide gel electrophoresis (2-DE) and electroblotting onto polyvinylidene difluoride membranes has been employed to rapidly isolate a number of proteins from a crude cell extract of a human colon carcinoma cell line (LIM 1863). The immobilized proteins were located by staining with Coomassie Brilliant Blue R-250, and selected protein spots were excised and subjected to Edman degradation. Our results demonstrate that overall sequence yields in the 3-20 pmol range can be achieved on protein spots from four identical 2-DE gels; approximately 150-200 micrograms of total protein was applied to a single 2-DE gel. An approximate two-fold increase in sensitivity of phenylthiohydantoin-amino acid detection (subpicomole range) was achieved by fitting our commercial sequencers with a simple sample transfer device which permitted the analysis of the total phenylthiohydantoin-amino acid derivative. N-Terminal amino acid sequence data was obtained for thirteen electroblotted proteins. All of these sequences positively matched those of proteins of known structure listed in the available protein sequence databases. Approximately 40% of the electroblotted proteins did not yield N-terminal sequence information, presumably because they had blocked N-termini (either naturally or artifactually). Internal amino acid sequence information was obtained from three proteins isolated by preparative 2-DE. This was achieved by in situ digestion of the proteins in the gel matrix with Staphylococcus aureus V8 protease, electrophoresis of the generated peptides in a one-dimensional gel, electrotransfer of the peptides to a polyvinylidene difluoride membrane and microsequence analysis of the electroblotted peptides.     

Microheterogeneity of apolipoprotein D as revealed by electroblotting following isoelectric focusing in Immobiline DryPlates.

The microheterogeneity of apolipoprotein D was examined by a procedure involving, in sequences: (i) electrophoresis in an immobilized pH 4-7 gradient in an Immobiline DryPlate-polyacrylamide gel supplemented with Ampholine pH 5-7, (ii) covering of the gel with sodium dodecyl sulfate-containing agarose, (iii) electroblotting onto a polyvinylidene difluoride membrane and (iv) immunological identification. Seven isoforms were obtained with partially purified apolipoprotein D. Using this technique the apparent pI values at 15 degrees C for the isoforms were 4.57, 4.67, 4.78, 4.83 and 5.95, 6.06 and 6.19 (SD +/- 0.05 for all). Direct staining of the Immobiline DryPlate could not reveal the isoforms of partially purified apolipoprotein D.     

Electrophoretically driven SDS removal and protein fractionation in the shotgun analysis of membrane proteomes

SDS is mostly used to enhance the solubilization and extraction of membrane proteins due to its strong detergency and low cost. Nevertheless, SDS interferes with the subsequent procedures and needs to be removed from the samples. In this work, a special gradient gel electrophoresis (GGE) system was developed to remove SDS from the SDS-solubilized protein samples. As a proof-of-principle experiment, the GGE system was designed to be composed of an agarose loading layer, six polyacrylamide fractionation layers with different concentrations and a high-concentration polyacrylamide sealing layer. The advantages of the GGE system are that it not only can electrophoretically remove SDS efficiently so that the protein loss resulted from the repeated gel washing after electrophoresis was avoided, but also can reduce the complexity of the sample, prevent the precipitation of proteins after loading and avoid the loss of proteins with low molecular weight during the electrophoresis. Using GGE system, about 85% of SDS in the sample and gel was electrophoretically removed and the proteins were fractionated. Compared with the two representative gel-based sample cleanup methods reported in literature, GGE-based strategy significantly improved the identification efficiency of proteins in terms of the number and coverage of the identified proteins.     

A technique for detecting antifungal activity of proteins separated by polyacrylamide gel electrophoresis

A technique was developed for the detection of antifungal activity of proteins after discontinuous polyacrylamide gel electrophoresis under native conditions. The antifungal activity is detected as growth inhibition zones in a homogeneous fungal lawn, grown in an agar layer spread on top of the polyacrylamide gel. The position of proteins with antifungal activity can be determined on a diffusion blot prepared from the same gel. The technique is illustrated for three antifungal plant proteins, i.e. alpha-purothionin, Urtica dioica agglutinin, and tobacco chitinase.     

One-step electroblotting of 2- to 100-kDa proteins onto a PVDF membrane

This article describes a method for electroblotting peptides and small proteins (< 100 kDa) from tricine gels onto a PVDF membrane. The major potential problem with these types of procedures is that proteins tend to stay in the gel under conditions where peptides are effectively eluted. The suggested protocol allows the complete transfer and binding of proteins and peptides in the range of 2–97 kDa.     

India ink staining after sodium dodecyl sulfate polyacrylamide gel electrophoresis and in conjunction with Western blots for peptide mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We present an approach that allows matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) peptide mapping of proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose (NC). After blocking the nitrocellulose membrane with polyvinylpyrrolidone-40 the immobilized proteins are visualized using India Ink staining which allows the detection of low nanogram amounts of protein. The utilization of a low concentration of Tween 20 (0.05%) in the India Ink staining solution does not negatively impair the quality of the mass spectra. Due to the virtual nondestructive nature of the stain proteolytic peptides could be recovered from the NC membrane. Taking into account minor precautions during the sample manipulation and concentration and by loading the sample onto a pre-crystallized matrix layer, high quality mass spectral data were obtained on <100 femtomoles of protein loaded onto the gel. Finally, the use of India Ink in conjunction with Western blot analysis is also demonstrated. A rat plasma protein, characterized by Western blot as a covalently modified protein-drug compound, was subjected to peptide mapping and post source decay (PSD) sequencing of peptides. The zomepirac-modified protein was identified as the alpha-subunit of fibrinogen.     

Evaluation of membranes used for electroblotting of proteins for direct automated microsequencing

Protein purification and characterisation have been age-old problems for the biochemist. A new era has arisen with the advent of one- and two-dimensional gel electrophoresis and high sensitivity automated protein microsequencing. These two tools along with electroblotting have made it possible to separate and analyse complex protein mixtures. We studied six different membranes compatible with Edman degradation chemistry to determine their efficiencies at binding proteins electroblotted from one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Their overall blotting-sequencing properties were also evaluated. We found that the polyvinylidene difluoride-based membranes out-performed the glass-based and polypropylene-based membranes under our selected experimental conditions. The problems associated with electroblotting and microsequencing are discussed.     

Protein identification methods in proteomics

A combination of high-resolution two-dimensional (2-D) polyacrylamide gel electrophoresis, highly sensitive biological mass spectrometry, and the rapidly growing protein and DNA databases has paved the way for high-throughput proteomics. This review concentrates on protein identification. We first discuss the use of protein electroblotting and Edman sequencing as tools for de novo sequencing and protein identification. In the second part, we highlight matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) as one of the main contemporary analytical methods for linking gel-separated proteins to entries in sequence databases. In this context we describe the two main MALDI-MS-based identification methods: (i) peptide mass fingerprinting, and (ii) post-source decay (PSD) analysis. In the last part, we briefly emphasize the importance of sample preparation for obtaining highly sensitive and high-quality MALDI-MS spectra.     

Determination of the Molecular Weight of Extracellular Ribonuclease Isoenzymes from Aspergillus Niger in Crude Extracts by Thin Layer Gel Filtration and Polyacrylamide Gel Electrophoresis

Abstract

The extracellular ribonucleases from Asperqillus niger culture medium were fractionated according to their molecular weight by thin layer gel filtration through Sephadex G100 superfine and the enzyme activity was detected by a standard staining technique on a replica print paper. Another replica paper was laid onto the top of a polyacrylamide gel and the absorbed proteins were separated by electrophoresis. By comparing the electrophoretic pattern with that of a control not subjected to gel filtration, the molecular weight of each isoenzyme in the crude extract could be determined. Gel electrophoresis however, is only used to establish the correspondence between the original electrophoretic pattern of the isoenzymes in the crude preparation and that detected on the replica print paper taken after the thin layer gel filtration run. There was good agreement between the values obtained for the crude and purified enzymes.     

Simultaneous detection of molecular weight and activity of adenylate kinases after electrophoretic separation

Adenylate kinases (AKs) are ubiquitous monomeric phosphotransferases catalyzing the reversible reaction, AMP + MgATP = ADP + MgADP, which plays a pivotal role in the energetic metabolism. In vertebrates, six AK isoforms are known. In this work, we report the detection of many AK isoforms directly on gel or NC after separation by denaturing electrophoresis and electroblotting, by an optimized protocol for the enzyme detection. The method allows to clarify the apparent MW of most of those AK isozymes that follow the cited reaction, especially onto NC where bands are sharper due to the absence of protein diffusion. In contrast, GTP:AMP phosphotransferases are not detectable. AK activity from many sources can be detected in both its reaction courses; ATP production appears as dark-blue bands, while ADP formation appears as nonfluorescent bands over a fluorescent background, under long-wavelength UV light. We show that nondenaturing gel electrophoresis is not the first choice for AK activity detection. Our method is different from the preceding reports on AK activity detection in bacteria after native polyacrylamide gel separations, in the absence of SDS or methanol. The procedure is also quantitative, allowing to determine the amount of enzyme present in samples.