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권무식 成均館大學校 科學技術硏究所 1992 論文集 Vol.43 No.2
Endo-B, the mouse form of Keratin 18, is the first member of the large intermediate filament gene family to be expressed during embryogenesis. Thus, the differentiation of cultured murine embryonal carcinoma cells has been used as a convenient model system of early mouse development. F9,22(mouse embryonal carcinoma) cells were treated with retinoic acid (5 x 10_6M/l) for 72 hours, and then they were fixed with the mixture of glutaraldehyde(2%) and formaldehyde(2%) in phosphate buffered saline for 5 min at 4℃. The fixed cells were immunoreacted with the rabbit anti-Endo-B antiserum. Endo-B was visualized by reaction with rhodamine conjugated goat anti-rabbit antibodies. Some of the differentiated F9 cells exhibited strong fluorescence indicating the expression of Endo-B filament.
미분화된 F9 세포에서 JunB에 의한 AP-1 활동의 유도
權戊植 成均館大學校 科學技術硏究所 1992 論文集 Vol.43 No.1
A full-length JunB cDNA was inserted into an eukaryotic expression vector, pHBAPr-l-neo(or, LK444), to construct a JunB cDNA expression plasmid, LK_4JunB. Undifferentiated murine embryonal carcinoma cells(F9 22 : 5 X 10 exp (6)/10ml) were cotransfected by the calcium phosphate method with DNAs of LK_4LAC(1.5㎍), 3 X TRECAT(3㎍) along with or without PvuII-cut LK_4JunB(8,㎍), in order to test a plausible role of the JunB or AP-1 activity. It was revealed that the cells cotransfected with the LK_4JunB showed some 20 times higher the CAT reporter gene activity than the value obtained from the cells without the LK_4JunB. This finding strongly suggest the fact that the JunB should have the AP-1 activity.
권무식 成均館大學校 科學技術硏究所 1990 論文集 Vol.41 No.1
A solid-phase electro-blot procedure was used for immunoassay of mammalian pyrurate dehydrogenase(E1) subunits a(Ela) and b(Elb). E1(porcine kidney) was fractionated by sodium-dodecyl sulfate polyacrylamide gel electrophoresis. Ela and Elb proteins were electrophoretically transferred onto nitrocellulose membrane with a constant current of 70mA as a function of time. The adsorbed proteins(Ela & Elb) were immunoreacted with rabbit anti-E1(porcine kidney) antiserum, then the immunoadsorbed Ela and Elb were identified by goat anti-rabbit IgG-horseradish peroxidase conjugate immunoassay kit. It has been found that the solid-phase immunoassay sensitivity of Ela and Elb increased in accordance with increment of transfer time up to a certain point under these experimenal conditions.
권무식,홍성렬 成均館大學校 科學技術硏究所 1990 論文集 Vol.41 No.1
Pyruvate dehydrogenase(El) composed of two nonidentical subunits a & b has been isolated from Pyruvate dehydrogenase complex(PDC) (porcine kidney). Essential steps in this purification process are involved in fractionation of PDC component by sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS -PAGE), electroelution of E1 (a, b) using dialysis membrane, and ethanol precipitation of eluate. Anti-El antiserum has been raised in a rabbit against El(a, b) purified from porcine kidney. The anti-El antiserum is immunoreacted with 20ng of Ela or Elb by a dot-blot assay.
權戊植 成均館大學校 科學技術硏究所 1987 論文集 Vol.38 No.2
Some datailed procedures for the isolation of biologically active poly(A^+) RNAs from the rat liver and in vitro synthesis of complementary DNA using the isolated poly(A^+) RNA as templates, were described.
3-oxoacid CoA-transferase cDNA 클론의 혈청학적 동정
權戊植 성균관대학교 기초과학연구소 1987 論文集 Vol.38 No.2
A cDNA clone for 3-oxoacid CoA-transferase was identified in a λgtll expression gene bank with a monospecific polyclonal rabbit anti-rat 3-oxoacid CoA-transferase. The cDNA clone bank was constructed from poly(A^+) RNA isolated from rat(12 day-old) brains. The cDNA clone was subcloned into M13mp18 and used for further characterization.
웨스턴 전이 : 피루브산 탄산효소 (흰쥐 간) Pyruvate Carboxylase (Rat liver)
權戊植 成均館大學校 科學技術硏究所 1987 論文集 Vol.38 No.2
Pyruvate carboxylase isolated from the mammalian rat and mouse livers were subjected to sodiumdodecyl sulfate-polyacrylamide gel electrophoresis. The pyruavte carboxylase on the gel was electrically transferred to pure nitrocellulose membrane (average pore size : 0.2㎛). The enzyme (antigen) immobilized on the nitrocellulose membrane, was identified by a double antibody assy. The Bio-Rad ImmunBlot(Goat Anti Rabbit-Horse Radish Peroxidase Conjugate) Assay Kit (Bio Rad Co.) was employed here. The electroblot conditions were best optimized for the pyruvate carboxylase. The detailed procedures were discussed in the text.
프라스미드 DNA의 대량 분리법 연구 및 아가로스 겔 전기영동상의 고찰
權戊植 成均館大學校 科學技術硏究所 1988 論文集 Vol.39 No.1
A novel method for a large scale isolation of plasmid pBR322 from Escherichia coli HB101 has been developed. The plasmid has been characterized on agarose gel electrophoresis.
생쥐 배종양 세포에 있어서 tPA 유전자 발현에 관한 연구
權戊植 성균관대학교 생명과학자원연구소 1994 生命資源科學硏究 Vol.1 No.1
F9 murine embryonal carcinoma cells expresed gene for tissue plasminogen activator(tPA), upon differentiation in vitro induced by retinoic acid(5×10 exp(-6)M/1). A recombinant plasmid(pBTPA) capable of tPA sense and antisense RNAs was constructed, and used for the RNA protection assay for tPA gene transcript in the differentiated F9 cells.
네오마이신 인산기전위효소 유전자의 F-9 세포 內 형질전환
홍성렬,권무식 成均館大學校 科學技術硏究所 1992 論文集 Vol.42 No.2
쥐 배종양세포, F-9, 22에 aminoglycoside 계통의 항생제 G-418 저항성을 갖도록 형질전환 시키었다. 즉, aminoglyucoside phosphotransferase 유전자를 갖고 있는 포유동물세포 발현 백터 pHβ APr-1-neo를 F-9, 22 세포에 electrophoration 법을 이용하여 transfection시키었다. 이 형질전환된 세포를 여러차례 subcultrue한 다음 neo 유전자 저항 능력이 소멸되지 않고 아주 안정하게 유지됨을 보았다. neo유전자로 형질전환된 클론은 RNAse protection assay로 관찰하였으며, 이를 위하여 BSpMC1rp를 합성하였다. A Plasmid PH βAPr-1-neo containing phosphotransferase gene (neo) was introduced into murine embryonal carcinoma (EC) cells (F-9, 22) by electroporation (250 volts/cm, 10 millisecond).. Colonies resistant to the drug G-418 were selected as plausible clones transfected by the neo. Elevated levels of the neo in the putative clones were confirmed by RNAse protection experiments, one of which named "F-9/neo". A 241 bp fragment of neo cDNA (EcoRl-Narl segment of the plasmid pMClneoA) was ligated to generate BSpMC1rp for the neo riboprobe synthesis. Transcription of EcoRl cut BSpMC1rp in the presence of 32pGTP yielded a 384 by probe, of which 284 by were protected by endogenous neo mRNA in the F-9 /neo clone.