• 분석과학
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• 제8권2호
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• pp.117-124
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• 1995

지속적인 음주로 인하여 간경변증으로 사망한 human 간에서 ethanol에 의해서 유도되는 것으로 추측되는 hemoprotein들을 확인 및 부분정제하였다. 이 hemoprotein을 정제하기 위하여 Mohamed 등의 방법을 변형하여 단백질을 정제하였고, SDS-PAGE 및 spectrum 양상을 관찰하였다. Triton N-101을 처리한 crude extract를 준비하여 CO gas를 bubbling시킨 후 Octyl-Sepharose CL-4B column chromatography에서 0.06% Lubrol PX로 용출한 다음 0.25% Lubrol PX로 용출하였다(Fig. 2). 0.06% Lubrol PX로 용출한 active fraction을 Hydroxyapatite와 DEAE-Sephadex A-25 column으로 정제하였다(Fig. 3, 4). 정제한 단백질을 12.5% SDS-PAGE를 실시한 결과 분자량은 대조군으로 사용한 흰쥐 간에서 정제한 단백질의 분자량은 55 KDa와 52 KDa였고, 돌연사한 사람의 간에서 정제한 단백질의 분자량은 62 48KDa이며, 간경변증으로 사망한 사람의 간에서 정제한 단백질의 분자량은 54KDa였고(Fig. 5). Cytochrome P450 함량은 20.8nmol/mg protein이며 회수율은 약 4.1%이고, 이들의 최대흡수 파장은 446nm이었다(Fig. 6).

• Parasites, Hosts and Diseases
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• 제51권2호
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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.

• 한국미생물·생명공학회지
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• 제16권5호
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• pp.393-397
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• 1988

고도 호열성균의 Adenylate kinase가 phospho cellulose column의 adenosine-penta-phospho adenosine affinity elution으로부터 균일하게 정제되었다. 분자량은 SDS PAGE와 gel filtration으로부터 22,000의 단량체로 밝혀졌다. 효소반응의 최적온도는 8$0^{\circ}C$이였으며 정반응의 활성화 에너지는 22.4kcal/mole이었다. 본 효소는 6M guanidine-HCI에 활성을 잃지 않았으며 10$0^{\circ}C$에서 한시간에 75%의 활성을 유지하였다. AMP, ADP, ATP에 대한 Km치는 0.01mM, 0.017mM, 0.067mM이었다.

• 미생물학회지
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• 제31권2호
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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.

• 환경위생공학
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• 제12권2호
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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.

• 대한수의학회지
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• 제45권1호
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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.

• 한국미생물·생명공학회지
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• 제20권1호
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• pp.25-33
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• 1992

Serratia marcescens ATCC 25419를 질소원이 풍부한 BHI 배지에서 황산 암모늄 분별 침전을 시킨 후 DAEA-Sephacel chromatography, Phenyl-Sepharose hydrophobic chromatography, Sephacryl S-400 gel filtration, native gel elution을 거쳐 ALS isozyme Rf 0.83을 분리하였다. 분리한 ALS isozyme Rf 0.83의 native 형태는 gel filtration을 이용하여 분자량을 측정한 결과, 531,400이었고, SDS-PAGE를 수행한 결과 55,000의 large subunit와 38,900의 small subunit로 구성된 multimer임을 알 수 있었다.

• 한국식품과학회지
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• 제23권4호
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• pp.452-458
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• 1991

체다치즈 숙성기간 중 존재하는 저온성세균을 분리하여 단백질분해능이 우수한 균주를 선발하였고 이 선발된 균주가 생산하는 효소의 특성을 연구하였다. 체다치즈 숙성기간 중 미생물의 변화를 관찰한 결과 내냉성세균이 일정하게 유지되고 있었는데 그 중 200개의 균주를 1차 선발한 후 단백질분해능이 우수한 균주 3개를 분리, 동정한 결과 P. fluorescens 두 종과 A. denitrificans였다. 그 중 활력이 가장 높은 P. fluorescens 65가 생산하는 효소를 gel filtration에 의해 정제한 결과 $190{\sim}230ml$ elution volume에서 protease가 용출되었고 SDS-PAGE로 분석한 결과 분자량은 47,000인 것으로 나타났으며 아미노산 조성은 Glu(14.96%)와 Ser(13.83%)의 함량이 제일 높았고 Met, Trp은 존재하지 않았다. P. fluorescens 65의 성장곡선에 따른 효소활력을 실험한 결과 세포증식이 활발한 대수증식기에서 단백질분해효소를 많이 분비함이 나타났으며 pH는 변화가 없이 일정하였다. Extracellular protease 65의 반응 최적온도는 $45{\sim}50^{\circ}C$ 사이었으며 최적 pH는 6.0으로 나타났다.

• Parasites, Hosts and Diseases
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• 제45권4호
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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.

• 한국양식학회지
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• 제10권4호
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• pp.425-433
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• 1997

황해산 두족류 6종(참갑오징어, Sepia esculenta ; 쇠갑오징어, Sepiella japonica : 한치꼴뚜기, Loligo chinensis : 살오징어, Todarodes pacificus : 참꼴뚜기, Loligo beka : 낙지, Octopus minor)의 안구단백질을 추출하여 항원-항체 교차반응을 실시하였다. 쇠갑오징어, 살오징어와 낙지의 안구단백질로 항혈청(antiserum)을 제작하였으며 갑오징어목, 살오징어목 및 낙지목에 속하는 두족류 6종의 안구단백질을 항원(antigen)으로 사용하였다. 갑오징어목 속하는 쇠갑오징어 및 참갑오징어에서 항원-항체 반응 양상이 매우 뚜렷하였으며 종간 비교에 기준이 될 수 있고 또한 유의성있는 침강선을 볼 수 있었다. 또한 gel filtration chromatography 방법으로 살오징어의 안구단백질을 분획하여 crystallin을 분리하였으며, 주 분획을 SDS-PAGE 전기영동 방법으로 분자량을 추정해 본 결과 약 20-35 KDa 였다.