• Analytical Science and Technology
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• v.8 no.2
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• pp.117-124
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• 1995

To Purify hemoproteins showing from 218nm absorbance, crude liver extract of human with hepatocirrhosis was treated with Triton N-101. Hemoproteins were purified by modification of Mohamed's method. This crude extract was applied to Octyl-Sepharose CL-4B column and the step elution was performed with 0.06% Lubrol PX and 0.25% Lubrol PX. The absorption of effluents were examined at 418nm and two peaks were appeared(Fig. 2). Hemoproteins were purified from Hyydroxyapatite and DEAE-Sephadex A-25 columns which the first peak was applied to(Fig. 3, 4). In death with suddenly, purified hemoproteins with 62 and 45kDa were obtained from 12.5% SDS-PAGE. In death with hepatocirrhosis, purified hemoprotein with 54kDa was obtainded from 12.5% SDS-PAGE(Fig. 5). Cytochrome P450 was purified to a specific content of 20.8nmol/mg protein with a recovery of about 4.1%. Absorbance maximum of these hemoproteins were 446nm at UV spectruum(Fig. 6).

• Parasites, Hosts and Diseases
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• v.51 no.2
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• pp.177-182
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• 2013

The purpose of the present study was to evaluate the potential role of the 27-Kilodalton (KDa) antigen versus Fasciola gigantica adult worm regurge antigens in a DOT-Blot assay and to assess this assay as a practical tool for diagnosis fascioliasis in Egyptian patients. Fasciola gigantica antigen of an approximate molecular mass 27- (KDa) was obtained from adult worms by a simple elution SDS-PAGE. A Dot-Blot was developed comparatively to adult worm regurge antigens for the detection of specific antibodies from patients infected with F. gigantica in Egypt. Control sera were obtained from patients with other parasitic infections and healthy volunteers to assess the test and compare between the antigens. The sensitivity, specificity, positive and negative predictive values of Dot-Blot using the adult worm regurge were 80%, 90%, 94.1%, and 69.2% respectively, while those using 27-KDa were 100% which confirms the diagnostic potential of this antigen. All patients infected with Fasciola were positive, with cross reactivity reported with Schistosoma mansoni serum samples. This 27-KDa Dot-Blot assay showed to be a promising test which can be used for serodiagnosis of fascioliasis in Egyptian patients especially, those presenting with hepatic disease. It is specific, sensitive and easy to perform method for the rapid diagnosis particularly when more complex laboratory tests are unavailable.

• Microbiology and Biotechnology Letters
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• v.16 no.5
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• pp.393-397
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• 1988

The adenylate kinase was purified from an extreme thermophile by adenosine-pentaphospho-adenosine elution from phosphocellulose column. The molecular weight was estimated to be 22,000 by SDS-PAGE and gel filtration. The optimum temperature of the enzyme activity was 8$0^{\circ}C$ and the activation energy was given as 22.4 kcal/mole. The enzyme even showed full activity after incubation at 9$0^{\circ}C$ or in 6M guanidine-HCI at 3$0^{\circ}C$ and retained 75% of its original activity even after 1 hour at 10$0^{\circ}C$. The Michaelis constants of the enzymes for AMP, ADP, and ATP were 0.01mM, 0.017mM and 0.067mM, respectively.

• Korean Journal of Microbiology
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• v.31 no.2
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• pp.175-178
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• 1993

Alkaline .alpha.-amylase expressed in the transformant, Baciollus subtills MB809, containing alkaline amylase gene cloned from alkalophilic Bacillus sp. AL-8, was purified through for step separation processes. The purified alkaline .alpha.-amylase had molecular weight of app[roximately 59, 000 daltons on SDS-PAGE and Sephaex G-100 gel filtration. Amino acid sequence of terminal portion of the enzyme was analyzed with pure amylase eluted form the SDS-PAGE gel. N-terminal amino acid sequence of .alpha.-amylase was determined by the Edman degradation method and resulted in $NH_{2}$-ser-thr-ala-pro-ser-(ile)-lys-ala-gly-thr-(ile)-leu. For C-terminal amino acid sequencing, purified .alpha.-amylase was digested with carboxypuptidase A and B, and reverse-phase HPLC gradient elution system resulted in -thr-trp-pro-lys-COOH.

• Journal of environmental and Sanitary engineering
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• v.12 no.2
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• pp.59-64
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• 1997

For purification of a 70kDa toxic protein of mosquitocidal delta-endotoxin from B. thuringiensis strain H9B, immuno-affinity chromatography was performed. After separation of 70kDa toxic proteins from the delta-endotoxin of the strain H9B on SDS-PAGE, the 70kDa toxic protein was subcutaneously injected into rabbit for making a polyclonal antibody. A anti-70kDa toxic protein was purified by a column chromatography packed with protein A-sepharose 4B gels. The 70kDa toxic protein from delta-endotoxin of the strain H9B was also purified by an immuno-affinity chromatography packed with CNBr-activated sepharose 4B gels conjugated anti-70kDa toxic protein after elution with 1/10M citric acid-1/5M Na$_{2}$HPO$_{4}$ buffer(pH3.2) containing 0.5M NaCl. The 70kDa toxic protein was purified through only one step-separation system, was demonstrated by SDS-PAGE and immunoblot.

• Korean Journal of Veterinary Research
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• v.45 no.1
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• pp.105-111
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• 2005

Canine sarcoptic mite (Sarcoptes scabiei var. canis) is ectoparasite which burrow usually in the stratum corneum of the skin of dogs. Antigens from the burrowing mites induce humoral and cellmediated immune responses in the hosts. The effect of antigenecity induced by somatic antigens of house dust mite (Dermatophagoides pteronyssinus) isolated by continuous elution has been evaluated in canine sarcoptic mites infestation. Continuous elution was carried out in 7.5% SDS-PAGE to isolate proteins of common antigens from somatic antigens of house dust mite. These eluted proteins from somatic antigens of house dust mite were confirmed by Western blotting in 7.5% SDS-PAGE, and eluted proteins (65, 60 kDa) were isolated. To evaluate the antigenetic effect of eluted proteins, eight dogs were divided as 4 groups such as non-vaccinated and non-challenged control (Group I), challenged control (Group II), vaccinated (Group III), and vaccinatedandchallenged (Group IV) groups. Group II and IV were artificially infested canine sarcoptic mites. Group III and IV were immunized with eluted proteins (65, 60 kDa). At the 6th week of the vaccination, the antibody titers of Group of IV were statistically significant higher than those of Group II (p<0.05). And antibody titers of Group III were also statistically significant higher than those of Group I (p<0.05). From these result, it is possible to replace somatic antigens of canine sarcoptic mites with eluted proteins from somatic antigens of house dust mites in order to diagnose and prevent the canine sarcoptic mite infestations.

• Microbiology and Biotechnology Letters
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• v.20 no.1
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• pp.25-33
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• 1992

One acetolactate synthase isozyme which has Rf value of 0.83 on polyacrylamide gel electrophoresis was purified from Sewatia marcescens ATCC 25419 by ammonium sulfate fractionation, DEAE-Sephacel chromatography, Phenyl-Sepharose chromatography, Sephacryt S-400 gel filtration followed by native gel elution. The native molecular weight of the enzyme was determined to be 531,400 by gel filtration method, and SDS-polyacrylamide gel electrophoresis separated the native enzyme into two polypeptides having molecular sizes of 55,000 and 38,900 respectively. In kinetic parameters, $K_m$ value for pyruvate was 2.54 mM, and $V_{max}$ was 21.75 nmoie/min/mg. The enzyme showed maximal activity around pH 8.0 and optimal temperature of the acetolactate formation was $37^{\circ}C$. Feedback inhibition studies indicate that the purified enzyme is rather resistant to branched chain amino acids when compared with acetolactate synthase isozymes of plants or other enterobacteria.

• Korean Journal of Food Science and Technology
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• v.23 no.4
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• pp.452-458
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• 1991

Psychrotrophs producing protease were isolated during ripening periods of Cheddar cheese and one of them containing the highest protease activity was identified as Pseudomonas fluorescens 65. The extracelluar proteolytic enzyme was partially purified from P. fluorescens 65 through the Sephadex G-100 gel filtration. The protease was eluted between 190 ml and 230 ml of elution volume of sodium phosphate buffer. The purified protease showed a single band in SDS-PAGE and its molecular weight was 47,000. The composition of amino acid for the protease was determined and the most abundant amino acids were glutamic acid (14.96%) and serine (13.86%). The optimum temperature and pH for the activity was $45{\sim}50^{\circ}C$ and 6.0, respectively.

• Parasites, Hosts and Diseases
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• v.45 no.4
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• pp.287-293
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• 2007

The identification and characterization of antigens that elicit human T cell responses is an important step toward understanding of Leishmania major infection and ultimately in the development of a vaccine. Micropreparative SDS-PAGE followed by electro transfer to a PVDF membrane and elution of proteins from the PVDF, was used to separate 2 novel proteins from L. major promastigotes, which can induce antibodies of the IgG2a isotype in mice and also are recognized by antisera of recovered human cutaneous leishmaniasis subjects. Fractionation of the crude extract of L. major revealed that all detectable proteins of interest were present within the soluble Leishmania antigens (SLA). Quantitation of these proteins showed that their expression in promastigotes is relatively very low. Considering the molecular weight, immunoreactivity, chromatographic and electrophoretic behavior in reducing and non-reducing conditions, these proteins are probably 2 isoforms of a single protein. A digest of these proteins was resolved on Tricine-SDS-PAGE and immunoreactive fragments were identified by human sera. Two immunoreactive fragments (36.4 and 34.8 kDa) were only generated by endoproteinase Glu-C treatment. These immunoreactive fragments or their parent molecules may be ideal candidates for incorporation in a cocktail vaccine against cutaneous leishmaniasis.

• Journal of Aquaculture
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• v.10 no.4
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• pp.425-433
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• 1997

The isolated soluble eye proteins from six species (Sepia esculenta, Sepiella japonica, Loligo chinensis, Todarodes pacificus, Loligo beka and Ocotopus minor) of class Cephaloopoda were compared by crossed immunoelectrophoresis methods using antibodies directed against total soluble eye proteins antigens. It showed a distinct antigen-antibody reaction between the species of order Sepioidea and various antiserum of class Cephalopoda. Our results suggested that the common precipitation lines of Sepia and Loligo species which were different from that of Octopus minor. By which obtained from elution of gel filtration chromatography and 12.5% SDS-polyacrylamide gel electrophoresis, the molecular weight of Todarodes pacificus eye protein (crystallin) was determined in abut 20-35 KDa.