Highly-Purified Natural Rubber by Saponification of Latex: Analysis of Residual Proteins in Saponified Natural Rubber

2008 ◽  
Vol 81 (1) ◽  
pp. 121-137 ◽  
Author(s):  
Jintana Yunyongwattanakorn ◽  
Yasuyuki Tanaka ◽  
Jitladda Sakdapipanich ◽  
Voratep Wongsasutthikul
Keyword(s):  
Natural Rubber ◽  
Sodium Hydroxide ◽  
Room Temperature ◽  
Sodium Hydroxide Solution ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Aqueous Sodium Hydroxide ◽  
Enzyme Linked Immunosorbent Assay ◽  
Nitrogenous Compounds ◽  
Sds Page

Abstract Highly purified natural rubber (NR) was prepared by saponification of fresh latex (FL-latex) and preserved high-ammonia latex (HA-latex) in the presence of surfactant to reduce the residual proteins in resulting solid NR. Saponification of latex diluted to 30% DRC was carried out with 1–7% (w/v) sodium hydroxide at room temperature for 1–7 hr at 70 °C and coagulated with formic acid. The nitrogen content of NR obtained by coagulation of the saponified latex markedly decreased to less than 0.014% by centrifugation of the saponified latex or soaking the coagulum in aqueous sodium hydroxide solution. The nitrogenous compounds from saponified NR (SAP-NR) were extracted with sodium dodecyl sulphate (SDS) aqueous solution and subjected to SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis to check the molecular weight of extracts. The extract from SAP-NR and deproteinized NR by protease (DPNR) for comparison was subjected to the analysis of allergic protein by FIT Kit method, based on Enzyme-Linked Immunosorbent Assay (ELISA) method. No extractable protein was observed in SAP-NR, whereas DPNR contained 1.5 μg/ml proteins. The results from SDS-PAGE analysis and FIT Kit test demonstrated that NR free from allergic proteins is obtainable by saponification of FL-latex with 1.5% NaOH at 70 °C for 1 hr or at room temperature for 24 hr.

scholarly journals Comparison of the Proteosomes and Antigenicities of Secreted and Cellular Proteins Produced by Mycobacterium paratuberculosis

2006 ◽  
Vol 13 (10) ◽  
pp. 1155-1161 ◽  
Author(s):  
Donghee Cho ◽  
Michael T. Collins
Keyword(s):  
Solid Phase ◽  
Expression Profiles ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Infected Cattle ◽  
Mycobacterium Paratuberculosis ◽  
Serologic Tests ◽  
Sds Page

ABSTRACT The protein expression profiles and antigenicities of both culture filtrates (CF) and cellular extracts (CE) of Mycobacterium paratuberculosis were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), one-dimensional electrophoresis (1-DE) and 2-DE immunoblotting, and enzyme-linked immunosorbent assay (ELISA). The CF proteins were harvested from supernatants of stationary-phase liquid cultures and concentrated by size exclusion filtration. The CE proteins were extracted by mechanical disruption of cells using glass beads and a high-speed agitator. Analysis of SDS-PAGE gels showed that the majority of CF proteins had low molecular masses (<50 kDa), whereas CE protein mass ranged more evenly over a broader range up to 100 kDa. By 2-DE, CF proteins had a narrow array of pI values, with most being between pH 4.0 and 5.5; CE proteins spanned pI values from pH 4.0 to 7.0. The antigenicities of CF and CE proteins were first determined by 1-DE and 2-DE immunoblotting with serum from a cow naturally infected with M. paratuberculosis. The serum reacted strongly to more proteins in the CF than the CE. Sera from 444 infected and 412 uninfected cattle were tested by ELISA with CF and CE as solid-phase antigens. Receiver-operator characteristic curve analysis of the ELISA results showed a significantly greater area under the curve for CF compared to CE (P < 0.05). A high degree of variability in protein binding patterns was shown with 1-DE immunoblot analysis with 31 sera from M. paratuberculosis-infected cattle. Collectively, these results indicate that serologic tests for bovine paratuberculosis may be improved by using proteins derived from CF instead of CE. To maximize the diagnostic sensitivity of serologic tests, multiple proteins will be required. Even so, a CF ELISA may not be able to detect all M. paratuberculosis-infected cattle, in particular those in the early stages of infection that have yet to mount an antibody response.

scholarly journals Antivinculin antibodies in sera of patients with immune thrombocytopenia and in sera of normal subjects

Blood ◽  
1992 ◽  
Vol 79 (1) ◽  
pp. 161-168 ◽  
Author(s):  
Y Tomiyama ◽  
R Kekomaki ◽  
J McFarland ◽  
TJ Kunicki
Keyword(s):  
Immune Thrombocytopenia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Protein ◽  
Normal Subjects ◽  
Enzyme Linked Immunosorbent Assay ◽  
Two Dimensional ◽  
Platelet Protein ◽  
Sds Page ◽  
Significant Difference

Abstract We have characterized a 120-Kd antigen that frequently reacts with serum antibodies from patients with immune thrombocytopenia or normal subjects. Immunoblots made after two-dimensional nonreduced/reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional isoelectric focusing/SDS-PAGE demonstrated that this 120-Kd protein has the same molecular weight under nonreduced or reduced conditions, is not a surface protein, and has an isoelectric point (pl) of 6.4 to 6.5. From these data, one likely candidate is the intracellular platelet protein, vinculin. Monoclonal antivinculin antibody reacts with this 120-Kd protein, and purified human platelet vinculin is bound by antibodies that recognize the 120-Kd protein. Therefore, we conclude that this 120-Kd protein is identical to vinculin. Data obtained from a sensitive enzyme-linked immunosorbent assay demonstrate the presence of naturally occurring antivinculin antibodies in many normal sera. However, the incidence of antivinculin antibodies in patient sera (67%; 55 of 82 sera) is significantly (P less than .01) higher than that in normal sera (40%; 32 of 80 sera), and there is a significant difference (P less than .05) between the mean levels of antivinculin antibodies in patient and normal sera. Whereas the levels of these antibodies in patient and normal sera overlap, 2 of 82 sera from patients with thrombocytopenia express unusually high levels of such antibodies. The pathologic significance of these antibodies remains to be determined.

scholarly journals MPB70 and MPB83 as Indicators of Protein Localization in Mycobacterial Cells

1998 ◽  
Vol 66 (1) ◽  
pp. 289-296 ◽  
Author(s):  
Morten Harboe ◽  
Harald G. Wiker ◽  
Gunni Ulvund ◽  
Bent Lund-Pedersen ◽  
Åse Bengård Andersen ◽  
...  
Keyword(s):  
Protein Localization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Culture Fluid ◽  
Secreted Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sds Page ◽  
Shock Proteins ◽  
Triton X

ABSTRACT Culture fluids after growth of Mycobacterium bovis BCG on Sauton medium contain actively secreted proteins and proteins released by bacterial lysis. BCG culture fluids and sonicates ofMycobacterium tuberculosis and Mycobacterium paratuberculosis were tested after separation by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The localization of marker proteins was determined by enzyme-linked immunosorbent assay and Western blotting with selected monoclonal antibodies of known specificities. Soluble secreted proteins (MPB64 and proteins of the antigen 85 complex) and three heat shock proteins (DnaK, GroEL, and GroES) were recovered in a single peak after gel filtration, indicating their occurrence as a free monomer in the culture fluid and cytosol, respectively. Other constituents eluted in two distinct peaks during gel filtration. The first peak corresponded to the void volume, indicating complex formation between several proteins or attachment to lipids in the surface layer or the cytoplasmic membrane; the second peak corresponded to the expected monomer size indicated by SDS-PAGE under conditions that separate proteins from each other during sample preparation. The two-peak group contained constituents with known lipid contents, the 19- and 38-kDa lipoproteins and lipoarabinomannan. The 26-kDa form of MPB83 behaved similarly. After extraction with Triton X-114, these constituents entered into the detergent phase, confirming the lipoprotein nature of 26-kDa MPB83. The MPB83 molecule was shown to be available on the surface of BCG Tokyo bacilli for reaction with monoclonal antibody MBS43 by flow cytometry.

scholarly journals Detection of Serum Thermolabile β-2 Macroglycoprotein (Hakata Antigen) by Enzyme-Linked Immunosorbent Assay Using Polysaccharide Produced by Aerococcus viridans

2001 ◽  
Vol 8 (2) ◽  
pp. 454-459 ◽  
Author(s):  
Mitsushi Tsujimura ◽  
Chuzo Ishida ◽  
Yasuko Sagara ◽  
Takashi Miyazaki ◽  
Koichi Murakami ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gas Chromatography Mass Spectrometry ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Serum Samples ◽  
Aerococcus Viridans ◽  
Sds Page

ABSTRACT Although a serum thermolabile β-2 macroglycoprotein (TMG) may play a role in host defense as a lectin, little is known of its related physiological functions, mainly due to a lack of appropriate methods for tracing the functions of TMG. We identified a polysaccharide fromAerococcus viridans, PSA, which reacts with TMG, and based on this finding, we developed an enzyme-linked immunosorbent assay to trace the functions of TMG. Using ethanol precipitation and DEAE-Sepharose and Sephacryl S-400 column chromatographies, we isolated PSA from cultured medium of A. viridans, and it exhibited specific binding against TMG in blood samples. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the isolated PSA showed ladder bands that implied the existence of repeating units composed of d-glucose,N-acetyl-d-glucosamine, d-mannose, and d-xylose, as confirmed by gas chromatography-mass spectrometry. SDS-PAGE and immunochemical analysis, using rabbit anti-TMG antibody, showed that PSA specifically binds solely to intact serum TMG but not to TMG heated at 56°C for 30 min, a condition under which antigenicity is lost. TMG in serum samples bound to PSA in a dose-dependent manner, and this binding was clearly suppressed by addition of PSA. These observations indicate that PSA is a useful adsorbent to TMG and can be used to develop appropriate methods for tracing the functions of TMG.

An Autoclave Treatment Reduces the Solubility and Antigenicity of an Allergenic Protein Found in Buckwheat Flour

2012 ◽  
Vol 75 (6) ◽  
pp. 1172-1175 ◽  
Author(s):  
HIROYUKI TOMOTAKE ◽  
RIKIO YAMAZAKI ◽  
MASAYUKI YAMATO
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Protein Solubility ◽  
Sodium Dodecyl ◽  
Major Allergen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Allergenic Protein ◽  
Future Studies ◽  
Autoclave Treatment ◽  
Sds Page ◽  
Buckwheat Flour

The effects of an autoclave treatment of buckwheat flour on a 24-kDa allergenic protein were investigated by measuring reduction in solubility and antibody binding. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the intensity of the major bands, including that of the 24-kDa allergen, was reduced by the autoclave treatment. The protein solubility in buckwheat flour was variably decreased by the autoclave treatment. Enzyme-linked immunosorbent assay analysis using a monoclonal antibody specific for buckwheat 24-kDa protein showed that the reactivity of protein extracts (10 μg/ml) from buckwheat flour was lowered by the autoclave treatment. The autoclave treatment may reduce the major allergen content of buckwheat. Future studies will determine if autoclaving treatments affect the allergenicity of the 24-kDa buckwheat protein.

An immunohistochemical investigation of the adult stage of the equine parasite Strongylus vulgaris

1998 ◽  
Vol 72 (2) ◽  
pp. 159-166 ◽  
Author(s):  
M.S. Mobarak ◽  
M.F. Ryan
Keyword(s):  
Gel Electrophoresis ◽  
Adult Stage ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Buccal Capsule ◽  
Enzyme Linked Immunosorbent Assay ◽  
Secretory Product ◽  
Somatic Muscle ◽  
Strongylus Vulgaris ◽  
Sds Page

AbstractAdult Strongylus vulgaris, collected from the caecum of infected horses and embedded in paraplast using standard methods, were sectioned for immunohistochemistry (IHC) studies. Antibodies were raised in rabbit against the excretory–secretory product (ESP) and against two constituent protein bands (28–30 kDa). The use of sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA) and immunoblotting indicated the immunogenicity of ESP and of the subunits (28–30 kDa). In ELISA, both rabbit hyperimmune sera recognized the ESP and (28–30 kDa) bands consistently and strongly. Both hyperimmune sera recognized most ESP subunits (80, 60, 54, 42, 35, 30, 20 and 15 kDa) in immunoblots. IHC, using light microscopy, suggested that the following worm tissues reacted strongly and positively with both antisera: amphids, tooth core, intestine, excretory gland and ducts, and hypodermis. Thus, either these are antigen-producing tissues, or antigens sharing common epitopes occur in them. The following tissues reacted weakly: body cuticle, buccal capsule cuticle, oesophagus, and also somatic muscle (non-contractile portion) perhaps due to diffusion of antigen from adjacent tissues. Preimmune serum gave a negative reaction with most worm tissues.

scholarly journals Antivinculin antibodies in sera of patients with immune thrombocytopenia and in sera of normal subjects

Blood ◽  
1992 ◽  
Vol 79 (1) ◽  
pp. 161-168
Author(s):  
Y Tomiyama ◽  
R Kekomaki ◽  
J McFarland ◽  
TJ Kunicki
Keyword(s):  
Immune Thrombocytopenia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Protein ◽  
Normal Subjects ◽  
Enzyme Linked Immunosorbent Assay ◽  
Two Dimensional ◽  
Platelet Protein ◽  
Sds Page ◽  
Significant Difference

We have characterized a 120-Kd antigen that frequently reacts with serum antibodies from patients with immune thrombocytopenia or normal subjects. Immunoblots made after two-dimensional nonreduced/reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional isoelectric focusing/SDS-PAGE demonstrated that this 120-Kd protein has the same molecular weight under nonreduced or reduced conditions, is not a surface protein, and has an isoelectric point (pl) of 6.4 to 6.5. From these data, one likely candidate is the intracellular platelet protein, vinculin. Monoclonal antivinculin antibody reacts with this 120-Kd protein, and purified human platelet vinculin is bound by antibodies that recognize the 120-Kd protein. Therefore, we conclude that this 120-Kd protein is identical to vinculin. Data obtained from a sensitive enzyme-linked immunosorbent assay demonstrate the presence of naturally occurring antivinculin antibodies in many normal sera. However, the incidence of antivinculin antibodies in patient sera (67%; 55 of 82 sera) is significantly (P less than .01) higher than that in normal sera (40%; 32 of 80 sera), and there is a significant difference (P less than .05) between the mean levels of antivinculin antibodies in patient and normal sera. Whereas the levels of these antibodies in patient and normal sera overlap, 2 of 82 sera from patients with thrombocytopenia express unusually high levels of such antibodies. The pathologic significance of these antibodies remains to be determined.

Reactions of N-(2- and 4-methylsulfonylphenyl)hydroxylamines and 2-methylsulfonylnitrosobenzene in dilute aqueous sodium hydroxide solution

1970 ◽  
Vol 48 (9) ◽  
pp. 1404-1413 ◽  
Author(s):  
K. B. Shaw ◽  
R. M. Heggie ◽  
R. K. Miller
Keyword(s):  
Sodium Hydroxide ◽  
Room Temperature ◽  
Sodium Hydroxide Solution ◽  
Aqueous Sodium Hydroxide ◽  
Major Product ◽  
Quantitative Yield ◽  
Aqueous Sodium Hydroxide Solution ◽  
Aqueous Sodium ◽  
Hydroxide Solution ◽  
Dilute Sodium Hydroxide

When N-(2-methylsulfonylphenyl)hydroxylamine (4) was treated with dilute sodium hydroxide solution the major product was always 2,2′-di(methylsulfonyl)azoxybenzene (5). At room temperature other significant products were 2-hydroxy-2′-methylsulfonylazoxybenzene (6a) and 2-methylsulfonylnitrobenzene (2), while 6a was also formed at reflux together with small amounts of 2-hydroxy-2′-methylsulfonylazobenzene (8), 2-methylsulfonylaniline (7), 3-methylsulfonyl-3′-nitro-4-amino-4′-hydroxybiphenyl (3), and 2. The compounds 5, 6a, and 7 were also obtained when 2-methylsulfonylnitrosobenzene (9) was boiled with alkali. The decomposition of N-(4-methylsulfonylphenyl)hydroxylamine (16) in dilute alkali at room temperature gave a quantitative yield of 4,4′-di(methylsulfonyl)azoxybenzene (17) and at reflux, mixtures of 17 and 4,4′-di(methylsulfonyl)azobenzene (18) were obtained. The modes of formation of the various products from the two hydroxylamines are discussed.

scholarly journals A Preliminary Study on Cross-Reactivity of Heat-Treated Quail and Hen’s Egg White Proteins in Young Children

Nutrients ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 2172
Author(s):  
Jeongmin Lee ◽  
Purevsan Gantulga ◽  
Changhoon Lee ◽  
Kyunguk Jeong ◽  
Eunjoo Lee ◽  
...  
Keyword(s):  
Young Children ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Egg White ◽  
Cross Reactivity ◽  
Inhibition Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hen’S Egg ◽  
Sds Page ◽  
Elisa Inhibition

We investigated the effects of different types of heat treatments on hen’s egg white (HEw) and quail egg white (QEw) proteins and their cross-reactivity in young children. Crude extracts of raw and water-boiled HEw and QEw and commercially developed stone-baked HEw were prepared. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was then performed. Patients diagnosed with HEw allergy were enrolled, and pooled sera were tested with each extract in an enzyme-linked immunosorbent assay (ELISA)-inhibition test. A skin prick test (SPT) and oral food challenge (OFC) were also performed. The median age of 12 patients was 2.5 years. SDS-PAGE results revealed strongly stained bands for the ovomucoid of boiled HEw and QEw, while stone-baked HEw displayed remarkable changes for all protein fractions. In the ELISA-inhibition test, pre-incubation of the sera led to a profound decrease, moderate decrease, and minimal decrease in the amount of IgE binding to boiled and raw HEw, boiled and raw QEw, and stone-baked HEw proteins, respectively. SPTs and OFC demonstrated cross-reactivity values of 41.7% (5/12) and 16.7% (2/12) for boiled QEw and stone-baked HEw, respectively. We observed moderate cross-reactivity between QEw and HEw. Boiling had a limited effect on altering egg allergenicity. Commercially developed, stone-baked HEw can be an alternative food for children with HE allergy.

scholarly journals Identification of Variants of the High Plains virus Infecting Wheat in Kansas

Plant Disease ◽  
2009 ◽  
Vol 93 (12) ◽  
pp. 1265-1274 ◽  
Author(s):  
Dallas L. Seifers ◽  
T. J. Martin ◽  
Tom L. Harvey ◽  
S. Haber ◽  
O. Krokhin ◽  
...  
Keyword(s):  
Amino Acid ◽  
Coat Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Sequences ◽  
Enzyme Linked Immunosorbent Assay ◽  
High Plains ◽  
Protein Amino Acid ◽  
Sds Page ◽  
Virus Isolates

The properties of two virus isolates (U04-82 and U04-83) obtained from two wheat (Triticum aestivum) plants expressing mosaic symptoms were investigated using enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), time-of-flight mass spectrometry (TOFMS), and infection of wheat with resistance to Wheat streak mosaic virus (WSMV). The coat protein mass was estimated by SDS-PAGE as approximately 32 kDa for U04-82 and 30 kDa for U04-83. The amino acid sequence of the coat protein of U04-82 was 99.6 and 85.5% identical to two isolates, ABC58222 and TX96, respectively, of High Plains virus (HPV) described from Texas. U04-82 was transmitted by wheat curl mites and caused significant yield reductions in wheat resistant to WSMV. U04-83 was actually two distinct virus isolates whose capsid protein amino acid sequences were only 57 and 50% similar to that of TX96. Antiserum prepared to a synthetic peptide from the sequence of the U04-83 isolate recognized the two U04-83 isolates, but not the U04-82 isolate.

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