Large-pore Polyacrylamide Gels Research Articles

Extensive Analysis of the Cytoplasmic Proteome of Human Erythrocytes Using the Peptide Ligand Library Technology and Advanced Mass Spectrometry

The erythrocyte cytoplasmic proteome is composed of 98% hemoglobin; the remaining 2% is largely unexplored. Here we used a combinatorial library of hexameric peptides as a capturing agent to lower the signal of hemoglobin and amplify the signal of low to very low abundance proteins in the cytoplasm of human red blood cells (RBCs). Two types of hexapeptide library beads have been adopted: amino-terminal hexapeptide beads and beads in which the peptides have been further derivatized by carboxylation. The amplification of the signal of low abundance and suppression of the signal of high abundance species were fully demonstrated by two-dimensional gel maps and nano-LC-MSMS analysis. The effect of this new methodology on quantitative information also was explored. Moreover using this approach on an LTQ-Orbitrap mass spectrometer, we could identify with high confidence as many as 1578 proteins in the cytoplasmic fraction of a highly purified preparation of RBCs, allowing a deep exploration of the classical RBC pathways as well as the identification of unexpected minor proteins. In addition, we were able to detect the presence of eight different hemoglobin chains including embryonic and newly discovered globin chains. Thus, this extensive study provides a huge data set of proteins that are present in the RBC cytoplasm that may help to better understand the biology of this simplified cell and may open the way to further studies on blood pathologies using targeted approaches.

A new approach for on-line enrichment in electrophoresis of dilute protein solutions

A method is described for on-line enrichment/zone sharpening of a sample of negatively charged proteins (an analogous method for cationic proteins can be designed). The sample is applied on the top of a 5-mm thick layer of a neutral polyacrylamide gel which rests on another 5-mm thick, large-pore polyacrylamide gel which contains positively charged groups. The latter gel layer is attached to the neutral gel column, used for the electrophoretic separation of the proteins. When a voltage is applied the proteins start migrating and become electrostatically adsorbed at the top of the charged, large-pore gel layer (pH 5.4). With the upper electrode vessel filled with a buffer of a pH higher (pH 7.7) than that employed in the enrichment step and with a voltage between the electrodes, these enriched proteins are released (because the enrichment gel is non-charged at pH 7.7) with zone sharpening and migrate into the 5-cm long column (i.d. 5 mm) of a neutral, large-pore polyacrylamide gel for electrophoretic analysis. Upon the electrophoretic migration from the enrichment gel into the separation gel a second zone sharpening may occur, if the increase in pH from 5.4 to 7.7 in the separation gel is not close to momentary. By employing colored test proteins the efficiency of the enrichment step is visually illustrated by a picture. The principle of the concentration method described has been employed also in chromatographic experiments and can with appropriate modifications also be used in other electrophoretic methods, such as capillary electrophoresis.

Sequence-dependent bending in plasmid pUC19.

The circular permutation assay has been used to characterize sequence-dependent bending in plasmid pUC19 (2686 bp). Linear, permuted sequence isomers of pUC19 exhibit different mobilities in large-pore polyacrylamide gels, suggesting that the plasmid contains two sequence-dependent bends, one located at approximately 806 bp, close to the start site of transcription, and the other at approximately 2617 bp, near the promoter of the ampicillin resistance gene. The mobility patterns are independent of the buffer in which the gels are cast and run, independent of pH over the range 6.5-8.6, and independent of the presence or absence of various monovalent ions added to the buffer, suggesting that the maxima in the mobility patterns correspond to stable bends in the helix backbone, not anisometric flexibility. Although the mobility patterns are unchanged when Ba(++) or Zn(++) ions are added to the buffer, several of the sequence isomers exhibit multiple sharp sub-bands in solutions containing Mg(++) or Ca(++) ions, indicating the presence of multiple conformational isomers. In addition, the mobility maximum near the start site of transcription is shifted by approximately 100 bp in the presence of Ca(++) ions, suggesting that the location of the actual bend center is somewhat variable, depending on the ionic composition of the surrounding medium. The number of maxima observed in the mobility pattern can be increased by adding a bend from another plasmid, such as Litmus 28; the number of maxima can be decreased by removing one of the bends in pUC19 by restriction enzyme digestion and religation. Hence, the number of maxima in the mobility pattern is an accurate indication of the number of stable, sequence-dependent bends present in the plasmid. The combined results indicate that the circular permutation assay in large-pore polyacrylamide gels is a reliable method of detecting and analyzing sequence-dependent bends in kilobase-sized DNA molecules.

Spinodal decomposition driven microsegregation in polyacrylamide gels

We present small angle static light scattering measurements on unusually large pore polyacrylamide gels, grown in the presence of the preformed polymer polyethylene glycol. We show that the large pores of these gels are due to a micro-segregation process, caused by the competition between gelation and segregation in the two-polymer solution. The presence of crosslinks prevents the system from undergoing a macroscopic phase separation which would otherwise occur via spinodal decomposition. The growth of the domains is eventually stopped by the gelation process and this process is ultimately responsible for the gel pore size.

Intrinsic curvature of plasmid DNAs analyzed by polyacrylamide gel electrophoresis.

The electrophoretic mobility of two small DNA plasmids, pUC19 and Litmus 28, linerarized by digestion with a variety of single-cut restriction enzymes, has been studied. The permuted sequence isomers migrate with identical mobilities in agarose gels, as expected, but exhibit different mobilities in large-pore polyacrylamide gels, suggesting that the parent plasmids contain sequence-specific sites of curvature and/or anisotropic flexibility. Both plasmids contain apparent bend centers near their origin(s) of replication; pUC19 also has a major apparent bend center near the promoter of the ampicillin resistance gene. These apparent bend centers are observed under a variety of experimental conditions, suggesting that they correspond to sites of stable curvature in the parent plasmids. Both plasmids also contain minor bend centers that are observed under a sub-set of electrophoretic conditions and disappear when divalent cations are added to the solution, suggesting that these apparent bend centers may correspond to localized regions of variable flexibility.

Electric birefringence of kilobase-sized DNA molecules

Transient electric birefringence has been used to characterize the rotational diffusion of linear, circularly permuted pBR322 and SV40 DNA molecules. The birefringence relaxation times vary with the site of linearization, suggesting that the circularly permuted DNAs have different conformations in solution. The longest relaxation times are observed for DNA sequence isomers linearized at the major bend centers identified by gel electrophoresis 1 1 N.C. Stellwagen, Electrophoresis, 15 (1995) 691. . SV40 sequence isomers linearized at other locations have faster, but approximately equal, terminal relaxation times, suggesting that their free solution conformations are relatively independent of the location of the bend center within the sequence. By contrast, the terminal relaxation times of the various pBR322 sequence isomers vary approximately in accord with their electrophoretic mobilities in large-pore polyacrylamide gels, suggesting that the different mobilities may reflect real conformational differences between the sequence isomers.

Use of polyacrylamide gel electrophoresis to detect structural variations in kilobase-sized DNAs.

The electrophoresis of linear, kilobase-sized DNA molecules with permuted sequences has been studied in polyacrylamide and agarose gels. Plasmid pBR322, bacteriophage phi X174, and the SV40 minichromosome were each digested with a series of single-cut restriction enzymes. The linearized, permuted isomers of all three DNAs exhibit different mobilities in large-pore polyacrylamide gels, suggesting that all three DNAs contain sites of anisotropic, sequence-dependent curvature. Various experimental parameters such as acrylamide concentration, crosslinker ratio and buffer composition affect the magnitude of the observed differential mobilities. Band sharpness appears to be optimal in polyacrylamide gels containing 6.9-8.1%T and 0.5-1%C. Only small mobility differences are observed for the linearized, permuted sequence isomers in agarose gels.

Immunochemical detection of peptides and proteins on press-blots after direct tissue gel isoelectric focusing.

A sensitive method is described for the detection of tissue peptides and proteins. They are separated by tissue isoelectric focusing using thin large-pore polyacrylamide gels, containing detergent and dimethylformamide, and are fixed with either glutaraldehyde or formaldehyde in gelatin-coated nitrocellulose membranes using press-blotting. The fixed peptide and protein antigens are visualized by immunoperoxidase staining. The spectrum of fixed tissue constituents may also be used to test antiserum reactivity and specificity in immunocytochemical staining procedures. Isoelectric focusing of 2 microL homogenates of the neurointermediate lobe of the pituitary allowed the immunodetection of peptides and proteins of various sizes and the determination of isoelectric points. However, direct application onto gels of small pieces of frozen tissue sections, sliced in a cryostat, appeared to be more efficient. By direct tissue isoelectric focusing of brain tissue, peptides were effectively eluted and separated from sections up to 100 microns thickness. This allowed the detection of small peptides with a detection limit of approximately 10 pg/section.

Silver staining of high molecular weight proteins on large-pore polyacrylamide gels

A large-pore gel for electrophoresis in the presence of sodium dodecyl sulfate, composed of 2.55% polyacrylamide crosslinked with 2.75% methylenebisacrylamide, is described. This gel has a resolving power for very high molecular weight proteins and can be stained with silver. The gel is suitable for fractionation of factor VIII/von Willebrand factor directly from plasma samples. Visualization by silver staining revealed a series of covalently bound multimers with molecular weights of up to 8 × 10 6. The procedure described should be useful also for studies on other very high molecular weight proteins and nucleic acids.

Dispersity of human factor VIII - Von Willebrand factor

Human factor VIII — Von Willebrand factor purified by gel chromatography in the presence of dextran has been studied with large pore polyacrylamide gel electrophoresis designed for analysis of highmolecular-weight proteins. It was demonstrated that purified factor VIII-Von Willebrand factor is not a single homogeneous entity, but consists of a series of immunologically related polymers. The polydispersity of factor VIII-Von Willebrand factor was confirmed by an immunochemical variant of the reversal-electrophoresis spreading test. With this technique it could be visualized that also factor VIII in plasma and cryoprecipitate is polydisperse in nature. As a consequence, changes in the polymer distribution of factor VIII-Von Willebrand factor can be an alternative explanation for results of dissociation experiments and for abnormalities in various diseases as demonstrated by cross-immunoelectrophoresis.

Isolation and characterization of two threonine-poor apolipoproteins of human plasma high density lipoproteins

Two apolipoproteins that are minor components normally of human plasma lipoproteins were discovered. They comprise up to 25% or more of the apolipoproteins in high density lipoproteins of some individuals with lipoprotein abnormalities associated with certain metabolic diseases and in individuals treated with amphotericin B for coccidiomycosis infection. The new apolipoproteins are distinctly different in amino acid composition, isoelectric point, and electrophoretic mobility from other known apolipoproteins. The new apolipoproteins are similar in amino acid composition. Both are unusually low in threonine, poor in valine and leucine, and rich in aspartic acid, glycine, alanine, and phenylalanine. They are relatively rich in arginine, although not as rich as the arginine-rich apolipoprotein, and have higher isoelectric points than other apolipoproteins except apoC-I. The more abundant of the two new apolipoproteins, of pI approximately 6.5, exists mainly as a dimeric protein of about 40 000 daltons and is dissociated by mercaptoethanol in the presence of sodium dodecyl sulfate. The less abundant one, of pI approximately 6.0, appears to be about 10 000 daltons. The threonine-poor apolipoproteins are associated mainly with plasma high density lipoproteins, particularly the denser HDL/sub 3/ subfraction, but small amounts were found in the very low and intermediate density lipoproteins.more » The HDL/sub 3/ with high content of threonine-poor apolipoproteins were indistinguishable from the normal HDL/sub 3/ by agarose or large pore polyacrylamide gel electrophoresis or by analytical ultracentrifugation. The lipid moiety of the abnormal HDL was richer in triglycerides and similar in phospholipid content to that of normal HDL.« less

Studies on cell-coat macromolecules in normal and virus-transformed BHK 21/C13Cells

Cells have been grown in vitro in the presence of radioactive precursors; much of the glucosamine label is incorporated into the surface material, part of it is then spontaneously released into the medium, part is readily removed by an EDTA wash, and further amounts may be released by a mild trypsin treatment or by a period of chase. The material in the EDTA was from variously labelled cultures has been fractionated by Sephadex G-200 chromatography; the excluded peak has been analysed by Sepharose 6B and DEAE-cellulose chromatography and by electrophoresis on large pore polyacrylamide gels. Its sensitivity to pronase and hyaluronidase has been determined, as well as its sugar, lipids and amino acid composition. Cell-coat material from Polyoma and Rous sarcoma virus-transformed cell lines failed to show significant differences in chromatographic properties and in sugar and amino acid composition. Minor but reproducible differences were found in the polyacrylamide electrophoresis migration patterns, when comparing glucosamine-labelled materials.

Gel electrophoresis of proteoglycans and glycosaminoglycans on large-pore composite polyacrylamide-agarose gels

The electrophoresis of proteoglycans and glycosaminoglycans on large-pore polyacrylamide gels which contained agarose as a support medium is described. The average pore diameter of the gels was sufficient to allow penetration of all but the very largest proteoglycans of articular cartilage, making it possible to distinguish “aggregates” from disaggregated molecules. The relative sizes of proteoglycans could be assessed, using only microgram quantities on these gels, as the mobilities were inversely related to hydrodynamic size as determined by gel chromatography. The method was able to separate single from double chains of chondroitin sulfate. On the other hand, the relative mobilities of separate chains of different types of glycosaminoglycans were a function of their charge density. The method has been applied to a pathological condition in which changes in the sizes of proteoglycans were evident.

Effect of solution components on large-pore polyacrylamide gel formation

Effect of solution components on large-pore polyacrylamide gel formation

Polyacrylamide-isoelectric-focusing a new technique for the electrophoresis of proteins

In this new technique, proteins migrate through a pH gradient to equilibrium positions within a slab of large-pore polyacrylamide gel. The pH gradient is established, and maintained, by carrier ampholytes (‘Ampholines’, L.K.B. Instruments Ltd.) under the influence of the applied electric field. The technique offers the advantages (ease of sample application and high resolving-power dependent only upon protein charge) of existing procedures utilising the ‘isoelectric-focusing’ principle ( Hoch and Barr, 1955; Tuttle, 1956; Svensson, 1962; Vesterberg and Svensson, 1966) and, in addition, employs simple apparatus, conserves expensive carrier-ampholytes, is very resistant to convective mixing, and permits the simultaneous separation of several mixtures. The gels may be stained for protein, subjected to densitometric analysis and dried for storage. Examples have been described of the use of the technique for the resolution of complex protein mixtures, the separation of isoensymes, the determination of the isoelectric points of proteins and for preparative purposes. Since a preliminary account of this work was presented ( Leaback and Rutter, 1968), a note has appeared concerning a similar procedure conducted in polyacrylamide rods ( Dale and Latner, 1968).