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PubMed1993

Simultaneous electroelution of whole SDS-polyacrylamide gels for the direct cellular analysis of complex protein mixtures.

A novel procedure which allow the rapid screening of complex protein mixtures in cellular assays is described. A device has been developed which allows a convenient, simultaneous electroelution of separated proteins from whole SDS polyacrylamide gels into narrow chambers each containing single or a few protein bands. We have optimized the conditions of the procedure and have obtained an efficient removal of SDS, leading to non-toxic protein fractions in a physiological buffer suited for direct testing in cell cultures. The responses generated by stimulating lymphocytes with the purified products have been compared to the native protein and a corresponding preparation of protein transferred to nitrocellulose particles. The method was used to investigate murine T cell responses to secreted mycobacterial antigens during infection with M. tuberculosis. A immunodominant secreted protein fraction was purified in a semipreparative scale by the procedure and used to immunize mice. The specificity of and lymphokine production by T cells generated in these animals were investigated. The device developed has various applications and provides a tool for the possible identification of new T cell antigens of importance for protective immunity.

Animals↗
PubMed1994

Cell adhesion to proteins separated by lithium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto a polyvinylidene difluoride membrane: a new cell-blotting technique.

Cell blotting, although conceptually simple, has failed to achieve wide practical application. Described here is a new cell-blotting technique which involves cell adhesion to protein bands after separation by lithium dodecyl sulfate-polyacrylamide gel electrophoresis (LDS-PAGE) and blotting onto polyvinylidene difluoride (PVDF) membrane at 4 degrees C. Cell bands adherent on PVDF are detected using hematoxylin, or propidium iodide (PI) staining followed by viewing under ultraviolet (UV) light. The technique allows quick microscopic visualization of adherent cells composing the bands, without requiring clearing of the membrane. Representative cell adhesion proteins from different sources, i.e., plant lectins (e.g., phytohemagglutinin, PHA; concanavalin A, ConA; and wheat germ agglutinin, WGA); extracellular matrix (ECM) proteins; and integral membrane proteins (e.g., recombinant soluble vascular cell adhesion molecule-1, rs VCAM-1) were tested for cell binding by the new cell-blotting technique using human lymphoid progenitor (NALM-6) and myeloid progenitor (KG1a) cell lines. Cell adhesion proteins retained their adhesion function in all cases tested. Specificity of cell binding on PVDF blot was demonstrated by inhibition of cell adhesion to WGA protein bands using an appropriate sugar, i.e., N-acetyl D-glucosamine. The cell blotting assay was comparable in sensitivity to Coomassie blue staining of protein bands. The ability to conduct protein extraction, separation and blotting at low temperature avoids thermal denaturation, thereby preserving the adhesion properties of the proteins. The electrophoretic/blotting system has unique detergent removal/protein renaturation properties and the ability to preserve functionally active adhesion protein complexes. The cell-blotting technique described is sufficiently robust for routine application in the investigation of novel cell adhesion proteins.

Cell Adhesion↗
PubMed1991

A study of electrophoretic mobility of DNA in agarose and polyacrylamide gels.

The aim of this paper is to clarify the mechanism of gel electrophoresis of DNA under constant-field conditions. We have conducted a large number of experiments on double-stranded DNA varying in length between approximately 10 and approximately 50,000 base-pairs, in both agarose and polyacrylamide gels ranging from 0.5% to 12% concentration, and with electric field strengths ranging from 0.5 to 8 V/cm. We have made (logarithmic) plots of velocity against length of DNA for all of the various test conditions. At the left-hand side of these plots, all of the empirical curves have a unique, standard shape. When the curves are normalized so that their left-hand parts coincide, a second feature emerges in that, while for any given test the curve follows the "master curve" up to a certain point, it then "breaks away" and becomes horizontal. We describe these two patterns of behaviour as "regions 1 and 2", respectively. We find simple yet comprehensive empirical formulae that fit the observations in the two regions of behaviour: these express the velocity in terms of length of DNA, electric field strength and gel concentration. We then construct two separate theories for the two regions of behaviour. The first theory involves the statistics of motion of an object through a random array of gel obstacles, with the instantaneous speed depending on the number of obstacles with which the object is currently in contact. The second theory is based on the mechanical hypothesis (for which there is other, independent support) that the DNA moves through the gel by piling up against a barrier, which eventually breaks or deforms under the resulting force, thereby allowing the DNA to move on to the next barrier. The statistical theory is an adaptation of existing work, while the mechanical one is new. We also describe experiments on the migration of repeated-sequence, curved DNA with length up to 1500 base-pairs, and we discuss its behaviour in terms of our two theories. Our studies by electron microscopy are consistent with the view that this repeated-sequence DNA adopts a superhelical configuration. Finally, we show that a very wide range of observations may be understood clearly by means of our two theoretical schemes.

Base Sequence↗
PubMed1976

Polyacrylamide-gel disc electrophoresis of native cerebrospinal fluid proteins with special reference to immunoglobulins and some clinical applications.

Polyacrylamide-gel disc electrophoresis has been applied to the fractionation of CSF samples obtained from 2m control subjects (range of age 16 to 77 yr, mean 41.0 yr) and from 157 patients with various neurological diseases, with the purpose of evaluating the diagnostic significance of separated protein bands. It was found that the immunoglobulins, with some exceptions, were fractionated in the G-zone which was defined as an area between the origin and 0.57 in terms of relative migration based on transferrin (RT). Fourty-four percent of the whole Bence Jones protein bands which were examined were detected in the area of RT 0.70-0.90. The normal values of CSF protein fractions were (means+/-SD):Pre-alb-zone, 10.97+/-2.31%; Alb-zone, 40.74+/-5.72%; A1-zone, 5.25+/-0.72%; A2-zone, 8.23+/-1.24%; B1-zone, 10.82+/-2.18%. B2-zone, 7.94+/-1.93%; G-zone, 16.05+/-2.49%. The following significant results were obtained: (1) When the total protein concentration (TP) of CSF is increased: the percentage of the Pre-alb-zone is lowered and that of the A1- and G-zones is increased in diseases such as neurosyphilis, infectious meningitis, brain tumour, multiple sclerosis, intervertebral disc protrusion and cerebral infarction. The A2-zone was diminished in cerebral infarction, and a similar change was seen in brain tumour with in addition a decrease of the B2-zone; (2) When the TP was normal: the percentage of the G-zone was increased in multiple sclerosis, brain tumour and intervertebral disc protrusion. In the latter 2 disorders the A1-zone also increased. This fraction also increased in the cases of cerebral infarction, whose mean age was, however, much higher than that of the control subjects.

Adolescent↗
PubMed1981

The demonstration of new human brain-specific proteins by high-resolution two-dimensional polyacrylamide gel electrophoresis.

High-resolution multiple two-dimensional polyacrylamide gel electrophoresis (ISODALT) has been used to analyse soluble protein extracts from human brain and 12 other human organs. Approximately 200 protein gene products can be visualised on an electrophoretogram of soluble human brain proteins. By electrophoresing extracts of different human organs separately and mixed with brain extract, 8 proteins have been found which appear to be present in brain in concentrations at least 20 times greater than in any other organ. Four of these brain-specific proteins have been identified by co-electrophoresis with purified proteins as 14-3-2 protein, creatine kinase-BB isoenzyme, aldolase C4 isoenzyme, and 14-3-3 protein. The identities of the remaining 4 proteins are unknown.

Brain↗
PubMed1985

The cerebrospinal fluid transferrin/tau proteins. A study by two-dimensional polyacrylamide gel electrophoresis (2D) and agarose isoelectrofocusing (IEF) followed by double-antibody peroxidase labeling and avidin-biotin amplification.

The cerebrospinal fluid (CSF) transferrin/Tau proteins were studied by two-dimensional polyacrylamide gel electrophoresis (2D) followed by immunoblotting and by agarose isoelectrofocusing (IEF), and subsequent double immunofixation, peroxidase staining and Avidin-Biotin Complex (ABC) amplification. The pattern of the Tau protein was similar but not equal to that of the transferrin (Tf). When a genetic variant of Tf was present in the serum, the same variant was also observed in the corresponding CSF Tf and in the Tau fraction. After neuraminidase treatment, both serum and CSF Tf moved to the Tau position on IEF and 2D. On 2D, no desialized precursors of the Tau proteins were detected, whereas the Tf precursors were always detected. No synthesis of the Tau globulin in the brain can, therefore, be inferred. In CSF not treated with neuraminidase, Tf is the only sialoglycoprotein clearly desialized, showing that the Tau fraction cannot be generated by neuraminidase action at CSF level. In fact, the treatment of serum and CSF proteins with neuraminidase produced a clear shift in the isoelectric mobility of all sialoglycoproteins. We clearly demonstrate that the Tau globulin is the result of neuraminidase activity not located in the CSF compartment. We suggest that Tf could be desialized by the action of neuraminidase at the brain level and then be "washed" into the CSF. Brain utilization of Tf, meeting the brain iron requirement, seems likely.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

Absence of biochemical heterogeneity in McArdle's disease. A high resolution SDS-polyacrylamide gel electrophoresis study.

Muscle biopsy extracts from a series of 6 patients with McArdle's disease were investigated by analytical SDS-polyacrylamide gel electrophoresis, to establish the presence or absence of the myophosphorylase protein subunit. In 4 cases, the band corresponding to the myophosphorylase subunit was totally absent from the electrophoretic staining pattern, and in 2 cases was present, but with a greatly reduced staining intensity compared with control normal patients; thus in none of the cases of McArdle's disease investigated was there evidence for a myophosphorylase subunit band of comparable staining intensity to that found in control normal patients. This result contrasts with previously reported findings (Feit and Brooke 1976) which suggested that McArdle's disease exists in biochemically heterogeneous forms; in one form of the disease myophosphorylase being totally absent and in a second form present to a similar extent as normal, but in an inactive form. On the basis of the results reported in this paper, we would suggest that myophosphorylase deficiency is a single gene disorder characterized by the absence or marked reduction of the myophosphorylase protein.

Adolescent↗
PubMedDate not supplied

Enzyme typing of southern African isolates of Plasmodium falciparum by polyacrylamide gel electrophoresis.

Nineteen southern African isolates of Plasmodium falciparum were typed by polyacrylamide gel electrophoresis, using 5 enzymes (glucose phosphate isomerase, adenosine deaminase, lactate dehydrogenase, NADP-dependent glutamate dehydrogenase and 6-phosphogluconate dehydrogenase). Limited variation was found amongst the isolates and the frequencies of variants were similar to those of isolates from other parts of the world. Eight of the isolates contained 2 forms of glucose phosphate isomerase, indicating clonal heterogeneity. One of these 8 isolates also contained 2 forms of adenosine deaminase and another showed 2 forms of lactate dehydrogenase.

Africa, Southern↗
PubMed1983

Two-dimensional polyacrylamide gel electrophoresis of hymenoptera venom and venom sac extracts.

Hymenoptera venom and venom sac extracts were studied by two-dimensional polyacrylamide gel electrophoresis using non-equilibrium pH gradient electrophoresis in the first dimension and sodium sulfate electrophoresis in the second dimension. Pure Apis mellifera (honeybee) venom collected by electrical stimulation was resolved into five major and more than 20 minor components. Polistes (paper wasp), Vespula (yellow jacket), Dolichovespula (aerial hornet) and Vespa (old world hornet) venom sac extracts contained more than 40 components each. These results illustrate the complexity of the mixtures in current use for immunotherapy for stinging insect hypersensitivity. The degrees of similarity observed between the various aspects of Polistes and Vespula studied correlate with the phylogenetic classification of these species.

Animals↗
PubMed1989

High resolution of honey bee (Apis mellifera) venom peptides by propionic acid/urea polyacrylamide gel electrophoresis after ethanol precipitation.

A new and simple gel electrophoretic method is described which enables the protein and polypeptide components of bee venom to be resolved on a single gel. The electrophoretic method allows octapeptides to be resolved and species as small as decapeptides can be detected at high sensitivity using the Coomassie blue staining method without prior fixation. This has been achieved by replacing acetic acid by propionic acid in acid/urea polyacrylamide gels and by controlling the amount of TEMED catalyst for the polymerisation of high concentration gels in order to obtain a low effective pore size. We demonstrated the value of ethanol precipitation as a rapid and efficient desalting the fractionation technique and propose that it could be used in combination with gel filtration to purify many of the peptides to homogeneity.

Animals↗
PubMed1992

Molecular mass determination and assay of venom hyaluronidases by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

We describe a procedure for molecular mass determination of hyaluronidases present in animal venoms from different families. Hyaluronidases can be revealed, following their electrophoretic separation in sodium dodecyl sulfate-polyacrylamide gel containing hyaluronic acid, by incubating the gel in Triton X-100 to remove sodium dodecyl sulfate and restore in situ enzyme activity. This method allows the detection of as little as 0.025 turbidity-reducing units after 2 hr incubation. All the hyaluronidases from the analyzed invertebrate venoms had a mass below 50,000 and showed only one component, while those from vertebrate venoms were more than 60,000 and in many instances contained more than one form.

Animals↗
PubMed1985

Protein and glycoprotein abnormalities in platelets from human Chediak-Higashi syndrome: polyacrylamide gel electrophoretic study of platelets from five patients.

Polyacrylamide electrophoretic analysis of proteins and Tritium-labelled glycoproteins of the platelets from five patients with Chediak-Higashi Syndrome shows the existence of marked quantitative differences when compared to normal platelets. While the glycoprotein abnormalities are solely related to the plasma membrane, some of the abnormalities detected in the Coomasie blue pattern are probably representative of defects related to the dense bodies and the alpha-granules. Some of the abnormalities found may, in part, explain the variability of aggregatory responses described in these patients, as well as the marked tendency towards desaggregation exhibited by platelets from humans with the Chediak-Higashi Syndrome.

Blood Platelets↗
PubMed1991

Detection of carboxymethylmetallothionein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

A sensitive method for detecting metallothioneins (MTs) by using silver staining and autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of carboxymethylated MTs is described. Carboxymethylation of MTs is indispensable because it prevents their aggregation, thereby allowing each of them to appear as a single band using SDS-PAGE. Metallothioneins can be detected with a limit of nanogram levels per lane. This method can be applied to MTs induced in in vitro cultured cells and in in vivo tissues.

Animals↗
PubMed1993

Analysis of rat urine proteins and allergens by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting.

BACKGROUND: In rats, urine has been identified as a major source of the allergens that cause laboratory animal allergy, an important occupational health problem. METHODS AND RESULTS: Urinary proteins and allergens of Wistar rats were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Proteins excreted by male and female rats during puberty were similar and of low molecular weight. In adulthood, moderate increases in diffuse staining of 26 and 21 kd occurred in female urine. In males the 17 kd protein increased dramatically and the 23 and 21 kd proteins moderately. The urine excretion of high-molecular-weight proteins (75 to 63 kd) increased with age in males (females not studied). Immunoblot studies with six sera showed allergens in urine of male and female rats of all ages, three of which were present in all urine (75, 68, and 21 kd). Three allergens (17, 16, and 15 kd) in female urine may be allergenically similar to the 17 kd allergen in adult male urine. Adult male urine allergens were studied further with sera from 83 rat-hypersensitive subjects. Major allergens were identified at 23, 21, and 17 kd, and all sera had IgE to one or more of these proteins. Twenty-seven percent had IgE to 68 and 63 kd allergens. Minor allergens were identified at 75, 51, and 44 kd. CONCLUSIONS: Rat urine is an important source of the major allergens associated with rat hypersensitivity. Age and sex markedly influence the protein and allergenic constituents of rat urine.

Aging↗
PubMed1986

An evaluation of the polyacrylamide gel electrophoresis fractionation method for the production of Mycobacterium tuberculosis skin test preparations. I. Production, physiochemical characterization and serological analyses.

As a part of a cooperative inter-laboratory WHO supported project raw tuberculins were produced and purified protein derivative (PPD, 18.7 g protein) was prepared. Employing a multistage preparative polyacrylamide gel electrophoresis (PAGE) method the PPD was separated into four fractions corresponding to 15, 7, 4.75 and 3.5% gel concentrations. The PAGE procedure resulted in three lots of material--each representing 11 electrophoretic runs. Immunodiffusion analyses showed that the largest number of precipitinogens was found in the 15% fractions and that some precipitinogens cross-reacted with preparations of Mycobacterium bovis BCG, M. intracellulare, M. kansasii, M. smegmatis and M. vaccae.

Antibodies, Bacterial↗
PubMed1996

Bioactive heparin surfaces from derivatization of polyacrylamide-grafted LLDPE.

Primary amine groups were introduced into polyacrylamide-LLDPE films, using the Hofmann degradation synthesis. The Hofmann degradation was studied at room temperature using sodium hypochlorite and sodium hydroxide at different concentrations. Diazotized heparin was covalently bound to the grafted LLDPE film via the primary amine groups. Surfaces were analysed with ESCA, ATR-IR, chloride titration and Toluidine Blue. Evaluation of the biological activity of the heparinized surfaces was made by measuring the capacity for binding antithrombin (AT) and inhibition of the activated coagulation factor XII (FXIIa). The heparinized surfaces were able to bind up to 3 pmol cm-2 of AT in solution with ionic strengths of I = 0.15 and I = 0.40. No activation of the adsorbed FXII was detected.

Acrylic Resins↗
PubMed1982

Identification of Rho(D) antigen in polyacrylamide gels by an enzyme-linked immunoassay.

Human erythrocyte membranes were solubilized in sodium dodecyl sulfate at 100 degree C and subjected to polyacrylamide gel electrophoresis. The gels were sliced into segments and each segment was incubated with anti-Rho(D) IgG, washed, and then incubated with goat anti-human IgG covalently linked to alkaline phosphatase. Para-nitrophenyl phosphate was added to each slice and the absorbance of the solution surrounding each slice was measured at 405 nm. This technique demonstrated that the Rho (D) antigen is a protein with a mol, wt between 13, 000 and 30,000. This method should be applicable to the investigation of other membrane-bound antigens.

Electrophoresis, Polyacrylamide Gel↗
PubMed1979

Automated scanning of 14C on polyacrylamide gel.

Cylindrical polyacrylamide gels were automatically analyzed for radioactive 14C using a commercially available radiochromatogram scanner. This method was capable of detecting peaks of [14C]protein containing 50 000--1 000 000 cpm. Under optimal conditions, gels could be scanned at a rate of 1 cm per min, allowing for analysis of 20--30 gels per day.

Autoanalysis↗