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PubMed1980

Separation of radiolabelled protein from brain and spinal cord of spinal hemisected rats on SDS polyacrylamide slab gels.

After hemisection of the spinal cord in rats, alterations in amino acid incorporation into protein have been observed in brain and spinal cord. Proteins mediating these changes have been studied in the present experiment. Male, Long-Evans hooded rats were given either a laminectomy and dura incision (sham) at spinal segment T2, a left spinal cord hemisection, or no operative procedures. One hour prior to utilization at 1, 3 and 14 days postoperation, amimals were injected subcutaneously with 200 microCi of [3H]-L-lysine and 200 microCi of [3H]-L-amino acid mixture. Samples were prepared for electrophoresis and proteins separated on linear gradient SDS polyacrylamide slab gels (7.5-20% acrylamide). The Gel slabs were cut into slices and processed for scintillation counting. In right somatomotor cortex, there was evidence for a general stimulation of amino acid incorporation at one day postoperation in animals which received surgery. At other postoperation intervals, significant increases in proteins in the regions of 90-150,000, 50-65,000 and 35-45,000 molecular weight (MW) were observed. In spinal cord, significant increases in proteins in the regions of 55-65,000, 30-45,000 and 15,000 MW were observed over postoperation time in all animals receiving surgery compared to normals. A large, significant increase in the radioactivity of proteins in the region of 18-20,000 MW were observed in the spinal cord lesion site of spinal hemisected animals alone at three days postoperation. These results indicate that increases in amino acid incorporation into the brain and spinal cord of laminectomized and spinal hemisected animals observed previously are theresult of both specific and nonspecific changes in protein incorporation of precursors.

Amino Acids↗

PubMed2004

Production of lignocellulolytic enzymes by Trametes gallica and detection of polysaccharide hydrolase and laccase activities in polyacrylamide gels.

White rot fungus Trametes gallica was studied for the production of lignocellulolytic enzymes: cellulase, xylanase, laccase, manganese-dependent peroxidase (MnP), and lignin peroxidase (LiP). The results demonstrated that low-nitrogen (2.2 mM N) and surface stationary cultivation favored production of extracellular MnP. MnP activity reached 118.1 UL(-1) while T. gallica was grown in a low-nitrogen culture containing phenylalanine. However, laccase levels observed in high-nitrogen (22 mM N) agitated cultures were much greater than those seen in low-nitrogen. The N source experiments seemed to reveal that NH4+ plays an important role in inducing MnP and laccase of the fungus. Results showed that T. gallica produces a series of the lignocellulolytic enzymes, and needs high N to produce all the enzymes during solid-state fermentation of wheat straw. This paper also presents a modified zymogram procedure to detect xylanase and laccase of T. gallica in polyacrylamide gel. Xylanase in crude enzyme of T. gallica was displayed by contacting protein gel strips with xylan substrate gels and by staining with iodine. By immersing the protein gel strips in o-tolidine solution, the blue-green zones representing laccase activity were visualized against a colorless background.

Basidiomycota↗

PubMed1992

Gradient polyacrylamide gel electrophoresis for determination of molecular weights of heparin preparations and low-molecular-weight heparin derivatives.

The M(r) values of pharmaceutical heparins and low-molecular-weight (LMW) heparin derivatives were examined as part of a collaborative study to develop methods for their characterization. Standard methods of M(r) determination rely on gel permeation high-performance liquid chromatography (HPLC). We report the use of gradient polyacrylamide gel electrophoresis (PAGE) to determine the M(r) values of pharmaceutical heparins and LMW heparin derivatives. This approach offers certain advantages over the HPLC method. Gradient PAGE analysis was performed in parallel, on multiple samples, with the same standard curve. HPLC was performed serially. Gradient PAGE gave higher resolution than HPLC, and thus, a mixture of easily obtained standards was used in place of individual standards for the construction of a standard curve. Heparin and various LMW heparin samples were analyzed by both gradient PAGE and conventional gel permeation HPLC methods. The number-average M(r), weight-average M(r), and polydispersity were examined by both techniques and found to be similar. This study demonstrates that gradient PAGE analysis is a sensitive method for the determination of the M(r) values of heparin and LMW heparin.

Carbohydrate Sequence↗

PubMed2004

Characterization of polyacrylamide based monolithic columns.

Supermacroporous monolithic polyacrylamide (pAAm)-based columns have been prepared by radical cryo-copolymerization (copolymerization in the moderately frozen system) of acrylamide with functional co-monomer, allyl glycidyl ether (AGE), and cross-linker N,N'-methylene-bis-acrylamide (MBAAm) directly in glass columns (ID 10 mm). The monolithic columns have uniform supermacroporous sponge-like structure with interconnected supermacropores of pore size 5-100 microm. The monoliths can be dried and stored in the dry state. High mechanical stability of the monoliths allowed sterilization by autoclaving. Column-to-column reproducibility of pAAm-monoliths was demonstrated on 5 monolithic columns from different batches prepared under the same cryostructuration conditions.

Acetone↗

PubMed1989

A quantitative study of relaxation rate enhancement produced by iron oxide particles in polyacrylamide gels and tissue.

The proton relaxation rate enhancements produced by two types of iron oxide particles, M4125 and AM125 (Advanced Magnetics, Cambridge, MA) designed as potential MR contrast agents, have been measured in polyacrylamide gels and in liver and spleen. The organ uptakes, time courses, and biodistributions of these materials have been measured using radiolabeling. The relaxation rate enhancements produced by these particles have been related to the concentration of iron per gram material at different magnetic field strengths. The relaxation rates increase linearly as a function of concentration at field strengths varying from 0.15 to 7 T. The relaxation effects of these particles in tissue are significantly different from the behavior in vitro. These results suggest that several different mechanisms contribute to relaxation effects in the presence of iron oxide particles and they depend strongly on the way the particles are sequestered. Diffusion effects for a specified TE may be enhanced using gradient or single-spin echoes rather than multiple echoes. AM125 is significantly more effective as a relaxation agent than paramagnetic metal ions only at relatively high fields, while M4125 is less effective per unit concentration.

Acrylic Resins↗

PubMed1989

Amniotic fluid acetylcholinesterase measurements: comparing immunochemical and polyacrylamide gel techniques.

In the present study, a recently reported immunochemical technique for measuring acetylcholinesterase (AChE) in amniotic fluid utilizing the 4F19 antibody was compared with the widely utilized polyacrylamide gel technique to determine whether the immunochemical assay provided an advantage in separating unaffected pregnancies from those associated with open spina bifida (OSB) and open ventral wall defects (OVWD). The study included (1) 73 amniotic fluid samples from unaffected pregnancies [alpha-fetoprotein (AFP) less than 2 MoM] with no visible gel AChE band, (2) nine bloodstained samples from unaffected pregnancies (AFP 2.2-4.0 MoM) with visible gel AChE bands, (3) 18 samples associated with OSB (AFP 2.2-7.0 MoM) with visible gel AChE bands, and (4) 20 samples associated with OVWD (AFP 3.2-53.5 MoM) with visible gel AChE bands. The immunochemical assay produced ranges of measurements in the four respective categories as follows: (1) 2-60 arbitrary units (AU): (2) 14-69 AU, (3) 61-593 AU, and (4) 22-476 AU. Eight of the nine unaffected pregnancies with visible gel AChE bands had immunochemical measurements below the highest measurement for the samples with no visible AChE band (60 AU), as did five out of 20 OVWD pregnancies. Two of the OSB cases had values of 61 and 62 AU. These data indicate that the 4F19 specific monoclonal antibody to AChE is capable of distinguishing unaffected from affected pregnancies with reasonable reliability but that more work needs to be done to establish the extent of overlap between the unaffected and affected populations.

Acetylcholinesterase↗

PubMed2004

Proteomic analysis of melanoma-derived exosomes by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry.

Exosomes are 40-100 nm vesicles released by numerous cell types and are thought to have a variety of roles depending on their origin. Exosomes derived from antigen presenting cells have been shown to be capable of initiating immune responses in vivo and eradicating established tumours in murine models. Tumour-derived exosomes can be utilised as a source of tumour antigen for cross-priming to T-cells and are thus of interest for use in anti-tumour immunotherapy. Further exploration into the protein composition of exosomes may increase our understanding of their potential roles in vivo and this study has examined the proteome of exosomes purified from cell supernatants of the melanoma cell lines MeWo and SK-MEL-28. The vesicular nature and size (30-100 nm) of the purified exosomes was confirmed by electron microscopy and sucrose density gradient centrifugation. Western blotting demonstrated the absence of calnexin and cytochrome c, verifying the purity of the exosome preparations, as well as enrichment of MHC class I and the tumour-associated antigens Mart-1 and Mel-CAM. The two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) protein profiles of exosomes from the two cell lines were highly comparable and strikingly different from the profiles of the total cell lysates. Mass spectrometric sequencing identified proteins present in 49 protein spots in the exosome lysates. Several of these have been identified previously in exosomes but some are novel, including p120 catenin, radixin, and immunoglobulin superfamily member 8 (PGRL). Proteins present in whole-cell lysates that were significantly reduced or excluded from exosomes were also identified and included several mitochondrial and lysosomal proteins, again confirming the proposed endosomal origin of exosomes. This study presents a starting point for future more in-depth protein studies of tumour-derived exosomes which will aid the understanding of their biogenesis and targeting for use in anti-tumour immunotherapy protocols.

Antigens, Neoplasm↗

PubMed2005

Comparative analysis of protein aggregates by blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a three-dimensional geometry gel.

We describe the comparative analysis of protein aggregates by combining blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 3-D geometry gel for simultaneous processing of many samples. The first native electrophoresis step, separating the aggregates, is carried out for a series of samples in parallel lanes within a slab gel. This gel is then placed on the top surface of a cylindrical, 3-D geometry gel for the second denaturing electrophoresis step, separating the proteins composing the aggregates. The samples migrate parallel to the vertical axis of the gel cylinder. Data are acquired online by photodetection of laser-induced fluorescence during electrophoresis. For this purpose, the samples are fluorescently labeled within the slab gel after the first separation step. A 3-D geometry gel separates the equivalent of many conventional SDS slab gels represented by vertical layers in the 3-D gel body. In this way, many samples are analyzed in the same gel under identical conditions, improving comparability and resolution and making the process considerably more efficient. This novel technique allowed the identification of several aggregate classes of recombinant proteins expressed in bacteria. We observed that proteins preferentially bind to homolog polypeptides, but also seem to form a trapping mesh co-aggregating with other proteins. The aggregation pattern revealed by this technique supplements data obtained from standard two-dimensional gel electrophoresis analysis. We expect interesting applications, for instance in aggregate monitoring of clinical samples. It should be feasible to quickly gain a diagnostic picture during amyloid-related neurodegenerative disease development or to observe drug effects on protein aggregation.

Bacterial Proteins↗

PubMed2006

Mass spectrometric sequencing of endotoxin proteins of Bacillus thuringiensis ssp. konkukian extracted from polyacrylamide gels.

The amino acid sequences of the crystal proteins of Bacillus thuringiensis ssp. konkukian strain HL-47 are unknown. We used 1-D denaturing polyacrylamide electrophoresis, nano-ESI-Q-TOF-MS, and protein database searching to analyze these proteins. On SDS-PAGE gels, a preparation of purified crystal proteins exhibited 110, 102, 76, 55, 37, and 30 kDa protein bands. Immunoblotting of the gel with antiserum raised to this preparation revealed that four crystal proteins, of 110, 102, 55, and 37 kDa, reacted with the specific antiserum. The 102-kDa major protein reacted strongly. The other crystal proteins showed weak immunoreactivity. The 102 and 55 kDa proteins were analyzed by ESI-MS. The internal amino acid sequence of the 102-kDa major protein has similarity to the sequences of the surface layer protein of B. thuringiensis ssp. finitimus and B. anthracis. However, the internal amino acid sequences of the 55 kDa protein did not show any homology to proteins in the databases. Proteomic analysis of these proteins leads to the conclusion that the sequence data provided the protein databases of the crystal proteins of the konkukian ssp.

Amino Acid Sequence↗

PubMed2006

C-terminal sequencing method for proteins in polyacrylamide gel by the reaction of acetic anhydride.

A successive C-terminal amino acid truncation reaction with acetic anhydride was applied on proteins in polyacrylamide gel. Protein bands separated by conventional SDS-PAGE were excised, partially fixed in the gel with glutaraldehyde ethanol solution, dehydrated with ACN and subjected to the truncation reaction with acetic anhydride formamide solution. Pre-treatment of the gel with pyridine aqueous solution was found to enhance the truncation reaction yields. After the truncation reaction, the products were treated with an aqueous solution of dimethylaminoethanol to hydrolyze oxazolone rings at the C termini of the truncated products and O-acetylated products of serine, threonine and/or tyrosine. Several commercially available proteins of 10-40 kDa, as determined by SDS-PAGE, such as myoglobin, trypsin inhibitor, alpha-hemolysin, cytochrome c, chymotrypsin C chain, elastase, acylase and histone H4, were subjected to the C-terminal analysis. The truncated proteins were in-gel digested with trypsin and the extracted peptides were analyzed by MALDI-TOF MS, giving rise to a series of molecular mass ions of the C-terminal truncated fragments corresponding to the C-terminal amino acid sequence of the relevant protein.

Acetic Anhydrides↗

PubMed1989

Rapid analysis of a peptide by fast-atom bombardment mass spectrometry after polyacrylamide gel electrophoresis.

Gel electrophoresis has been a powerful technique for the separation of peptides and proteins for many years. After electrophoresis separation on a polyacrylamide gel, the peptide bradykinin was localized using Coomassie Blue as a staining dye. Excess dye was removed by washing the gel with water. For mass spectrometric analysis, bands containing the peptide were crushed, extracted with acetic acid and the eluent applied to the fast-atom bombardment probe. Under these conditions the protonated molecule of bradykinin was clearly observed. Also apparent were sequence ions at about the same intensity observed from authentic bradykinin.

Bradykinin↗

PubMed2002

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.

Amino Acid Sequence↗

PubMed1999

Chemical synthesis of biotinylated histones and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/streptavidin-peroxidase.

Recently, Hymes and co-workers demonstrated that human biotinidase (EC 3.5.1.12) specifically biotinylates histones, suggesting that biotin may have a specific role in transcription and replication of DNA. In the present study, we sought to biotinylate histones in vitro for later use as standards in the quantitation of histones biotinylated in vivo. We also sought to develop a procedure for electrophoretic separation and streptavidin-peroxidase detection of the various classes of biotinylated histones. Histones H1, H2a, H2b, H3, and H4 from calf thymus were biotinylated using sulfosuccinimidobiotin at pH 7.5. Stoichiometries of biotin/histone were determined either by 4'-hydroxyazobenzene-2-carboxylic acid/avidin assay or by avidin-binding assay. The stoichiometries of biotinylation (mol biotin/mol histone) were as follows: H1, 3.9 +/- 0.17; H2a, 1.7 +/- 0.11; H2b, 1.8 +/- 0.11; H3, 0.029 +/- 0.0012; H4, 0.006 +/- 0.0002. When two synthetic polypeptides were used as substrates for biotinylation, the stoichiometry of poly-l-lysine was 2.8 +/- 0.14 mol biotin/mol; in contrast, the stoichiometry of poly-l-arginine was less than 0.3 x 10(-3) mol biotin/mol. These data suggest that primary amino groups of histones biotinylated by sulfosuccinimidobiotin were lysine rather than arginine. Detection and identification of biotinylated histones were accomplished by electrophoretic separation on 16% polyacrylamide gels; the separated histones on nitrocellulose transblots of the gels were detected using streptavidin-peroxidase with 4-chloro-1-naphthol as the substrate. We conclude that sulfosuccinimidobiotin does biotinylate each of the five classes of histones and that the stoichiometry of biotinylation is sufficient for detection on nitrocellulose transblots by streptavidin-peroxidase.

Amidohydrolases↗

PubMed1993

Purification of cysteine-rich bioactive peptides from leukocytes by continuous acid-urea-polyacrylamide gel electrophoresis.

A new continuous acid-urea-polyacrylamide gel electrophoresis (CAU-PAGE) preparative method was developed and used to purify rabbit and human defensins. With it, we identified two post-translationally modified forms of rabbit defensins NP-1 and NP-2, and purified a processed RANTES (beta-intercrine) peptide from leukophoresed human leukocytes. CAU-PAGE afforded approximately 70% recovery of rabbit defensin NP-5. The recovered defensins were not N-terminally modified, and their in vitro antimicrobial activity was equivalent to that of defensins purified by previously described chromatographic methods. Since CAU-PAGE is performed under nonreducing conditions, it should be especially useful for purifying cationic peptides with intramolecular disulfide bonds, such as defensins and alpha or beta-intercrines.

Amino Acid Sequence↗

PubMed1993

Staining technique for phosphatases in polyacrylamide gels.

The described staining technique affords a means to detect and evaluate directly on the electrophoresis polyacrylamide gel the activity of phosphatases present in complex extracts or in purified solutions, in particular phosphatases requiring alkaline pH for optimum activity, e.g. fructose bisphosphatase, sedoheptulose bisphosphatase, and alkaline phosphatase, and 5'-nucleotidase. The method is based on a modified malachite green procedure determining orthophosphate released by the specific enzyme reactions carried out in the gel and leads to the formation of sharp blue-green-colored bands on a pale blue background. Staining intensity is proportional to the amount of Pi formed. The lower limit of phosphate detection was in the order of 0.2 nmol. This method provides a routine tool for comparative studies of biochemical properties and isozymic composition of phosphatases from diverse sources.

5'-Nucleotidase↗

PubMed1993

Renaturation of calcium/calmodulin-dependent protein kinase activity after electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels to membranes.

A method is described for analyzing calcium/calmodulin-dependent protein kinase activity in crude or purified samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to PVDF membrane. The blotted protein is denatured in situ with guanidine HCl and renatured in buffer containing NP-40. The membrane with bound protein is incubated with [gamma-32P]ATP and buffer containing the activators Ca2+ and calmodulin resulting in autophosphorylation of a subset of bound kinases. Two of the three major kinase activities detected in as little as 5 micrograms of crude brain or spinal cord homogenates are the alpha (M(r) = 50-52,000) and beta (M(r) = 58-62,000) isoforms of Ca2+/calmodulin-dependent protein kinase II. A third unidentified kinase of M(r) = 90-95,000 is not dependent on Ca2+ and calmodulin for activity. The membrane can be used for immunoblotting, phosphoamino acid analysis, or peptide mapping after the in situ renaturation and phosphorylation procedure. Detection of kinase activity in this assay is dependent on autophosphorylation of the enzyme. Therefore another procedure is described in which the blotted proteins are denatured and renatured in situ and assayed by measuring incorporation of phosphate into an exogenous peptide substrate specific for calcium/calmodulin-dependent protein kinase II.

Amino Acid Sequence↗

PubMed1993

Nonurea sodium dodecyl sulfate-polyacrylamide gel electrophoresis with high-molarity buffers for the separation of proteins and peptides.

A sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a discontinuous buffer system for separation of both peptides and proteins, which is a modification of the Laemmli system, is described. In the modified procedure, twofold higher concentrations of buffers in the separating gel and the running buffer solution allow superior resolution for peptides as small as M(r) 5000. The resolution of peptides was dependent on salt concentrations in the systems in which sodium chloride was partially substituted for Tris-HCl buffer and buffer concentrations were varied. In the stacking gel of the modified procedure, detection of peptides and SDS demonstrated a sharp stack of peptides at the trailing edge of the SDS stack. On the other hand, this SDS stack included peptides, forced them to diffuse, and produced a broad starting zone under Laemmli conditions. In addition, following expansion of the SDS stack impaired peptide resolution further in the separating gel. Accordingly, the different interaction with the SDS stack in the stacking process was found to produce different resolution of peptides in the electrophoretic procedures. The modified conditions have potential to provide a superior alternative to the Laemmli system for analysis of various proteins.

Buffers↗

PubMed1993

Multiple bands on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of proteins due to intermolecular disulfide cross-linking.

Gel electrophoresis has been used extensively as an analytical technique to check the purity and to determine the molecular weight of proteins. Improved levels of detection of proteins by silver staining of the gels have made the technique more sensitive in detecting heterogeneity. We report here some interesting observations about the anomalous behavior of some proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This report is concerned with the appearance of multiple bands, after silver staining, on the SDS-PAGE gels of proteins which are shown by HPLC to be homogeneous. We also show the reasons for the appearance of bands that originate from 2-mercaptoethanol itself.

Cross-Linking Reagents↗