• Preventive Nutrition and Food Science
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• 제7권1호
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• pp.43-47
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• 2002

Crude caseinomacropeptide (CMP) was prepared from Na-caseinate using a commercial renneting enzyme. Most of the crude CMP was released from the Na-caseinate by hydrolyzing with the enzyme for 40 min. The hydrolysis of the k-casein with carbohydrate was slower than that of the k-casein without carbohydrate, as shown by the analyses of the sialic acid content and the tricine-SDS-polyacrylamide gel electrophoresis. The yield of crude CMP from Na-caseinate was 3.7%. Cation exchange chromatography showed that the crude CMP consisted of 40.5% CMP and 59.5% caseinogylcomacropetide (CGP). The effect of the TCA concentration on the solubility of CMP and CGP was determined by using crude CMP. The amounts of crude CMP and sialic acid decreased in the proportion to the increase of trichloroacetic acid (TCA) concentration from 2 to 12%, suggesting that the CGP containing carbohydrate, as well as the CMP having no carbohydrate, was precipitated in a range of 4 to 12%, depending on the TCA concentration. This result supports the hypothesis that the different non-glycosylated and glycosylated forms of CMP have different sensitivities to TCA precipitation.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권3호
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• pp.215-222
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• 2006

The Lowry method was used in this study to measure protein in Haemophilus influenzae type b (Hib) conjugate vaccines (polyribosylibitol phosphate-tetanus toxoid; PRP-TT) using deoxycholic acid (DOC) to induce protein precipitation. Trichloroacetic acid (TCA) did not induce precipitation adequately from the Hib conjugate bulk and the freeze-dried Hib conjugate product. Its yield was approximately 50%. The matrix structure of Hib conjugate inhibits precipitation by TCA. Although the Lowry method can be carried out without precipitation in Hib conjugate bulk when no residual impurities (adipic acid dihydrazide [ADH], 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide-HCI [EDAC], phenol and cyanogens bromide [CNBr], etc.) are present, it cannot be used for Hib conjugate products that contain sucrose 8.5%, because 8.5% concentration of sucrose enhanced the protein concentration. DOC- and HCl-induced precipitation is an alternative method for evaluating the protein content of the Hib conjugate bulk and the Hib conjugate product. The precipitation was optimal with a final concentrate of 0.1% for DOC at $4^{\circ}C$ and pH 2. This Lowry method, using DOC/HCI precipitation to induce protein precipitation, was confirmed a consistent, reproducible, and valid test for proteins in Hib conjugate bulk and its freeze-dried product.

• Journal of Applied Biological Chemistry
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• 제51권3호
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• pp.88-94
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• 2008

Three different protein extraction methods-phenol extraction, trichloroacetic acid (TCA) precipitation, and desalting/TCA precipitation-were compared to determine the optimal reproducible high resolution 2-dimensional (2-D) electrophoresis for each chungkugjang, doenjang, and kochujang samples. The soluble proteins from Chungkugjang extracted by phenol were separated with high reproducibility and resolution, and gained 1.75- to 3-fold more protein spots on 2-D gel than those from the other methods. On the contrary, the extracted proteins from doenjang and kochujang treated by desalting/TCA precipitation method showed about 1.5- to 3.3-fold more protein spots on 2-D gel. Using the established methods, the changes in the protein profiles of the fermented soy foods were monitored during the fermentation period by 2-DE. One of the major proteins in soy, $\beta$-conglycinin $\alpha$-subuint, and some proteins with unknown functions were localized on 2-D gel as the protease-resistant proteins throughout the fermentation period of doenjang. Changes in the protein profile monitored by the established methods can provide basic information on unfolding the mechanisms of the generation of biofunctional activity in the fermented soy foods.

• 한국식품영양과학회지
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• 제27권6호
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• pp.1117-1124
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• 1998

Chymosin was purified from commercial rennet with DEAE Sepharose CL 6B and immobilized on CeliteTM using glutaraldehyde. Whole casein from fresh raw milk was hydrolyzed by immobilized chymosin and total CMP was isolated by trichloroacetic acid(TCA) and ultrafiltration, and characterized. The amount of chymosin purified from 15g commercial rennet by DEAE Sepharose CL 6B was 0.16g and 18mg of chymosin was immobilized on 1g of CeliteTM by 5% glutaraldehyde. Immobilized chy mosin hydrolyzed most of casein on whole casein within 2 hours to leave para casein and casei nomacropeptide(CMP). The total CMP isolated from 10g of whole casein hydrolyzate by TCA and ultrafiltration was 0.4g and 0.1g, respectively. Results of electrophoresis, amount of sialic acid, com position of amino acid and ratio of A280 to A214 showed that total CMP by TCA was purer and had more CMP without carbohydrate than one by ultrafiltration. CMP isolated from total CMP by 12% TCA precipitation was 50% of total CMP and most of caseinoglycopeptide(CGP) was removed from total CMP, indicating less amount of sialic acid in CMP than in total CMP.

• 한국식품영양과학회지
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• 제9권1호
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• pp.15-22
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• 1980

자원(資源)이 풍부한 적이용해조(赤利用海藻)를 단백질(蛋白質) 자원(資源)으로 이용(利用)하기 위(爲)하여 지충이, 잘피 및 잘피 등(等) 2종(種)의 해조(海藻)와 1종(種)의 해산식물(海産植物)을 선정(選定)하여 이의 수용성(水溶性) 단백질(蛋白質) 추출조건(抽出條件)을 검토(檢討)하였다. 1) 첨가수량(添加水量)은 곰피는 1:150(W/V)의 비(比)로 가(加)했을 때 추출효과(抽出效果)가 좋았고, 잘피와 지충이는 1:100(W/V)일 때가 좋았다. 2) 추출시간(抽出時間)은 총질소(總窒素)의 경우 2시간(時間) 이상(以上)에서, TCA 불용성(不溶性) 단백질(蛋白質)은 1시간(時間) 정도(程度)가 효과적(效果的)이었다. 3) 추출온도(抽出溫度)는 $40-50^{\circ}C$에서 추출성적(抽出成績)이 좋았으며 $70^{\circ}C$ 이상에서 약간 높아지나 TCA 불용성(不溶性) 질소(窒素)는 증가하지 않으므로 고온추출(高溫抽出)은 불리(不利)하였다. 4) pH의 영향(影響)은 전시료(全試料)가 pH 9 이상(以上)에서 높은 추출율(抽出率)을 보였으나 TCA 불용성(不溶性) 질소(窒素)는 pH 7 이상(以上)에서는 증가(增加)하지 않는 관계로 pH $6{\sim}7$에서 추출(抽出)함이 유리(有利)하였다. 5) 순단백질(純蛋白質) 침전(沈澱) 분리(分離)는 pH 조절법(調節法)에 의(依)한 처리(處理)가 질소(窒素) 함량(含量)이 가장 높았다. 6) 추출단백질(抽出蛋白質)을 정제(精製)함으로써 아미노태질소(態窒素)의 함량(含量) 및 총질소(總窒素)에 대(對)한 백분율(百分率)도 높일 수 있는 효과(效果)를 얻을 수 있었다.

• Applied Biological Chemistry
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• 제39권2호
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• pp.123-126
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• 1996

도축동물의 대부분 폐기되는 혈액으로부터 기능성 소재를 발굴하고 수질오염을 방지하기 위해서 소, 돼지 도축혈액으로부터 간단하고 신속한 방법으로 혈장 단백질을 분리하는 방법을 개발했고 그것을 혈분접착제로 응용하였다. 혈액을 원심분리한후 혈장단백질은 2% TCA 또는 0.6N HCl을 최종농도가 되게 첨가하여 침전시킴으로써 얻어졌다. 소, 돼지 혈장단백질을 비교 분석하기 위하여 SDA-PAGE를 하였고 얻어진 혈장단백질을 이용하여 혈분접착제로서 합판제조에 사용하여 다른 합성접착제와 접착성 시험을 한 결과 다른 합성 접착제와 비교해서 만족한 결과를 얻었다.

• Molecules and Cells
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• 제22권1호
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• pp.119-125
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• 2006

The rat is an accepted model for studying human psychiatric/neurological disorders. We provide a protocol for total soluble protein extraction using trichloroacetic acid/acetone (TCA/A) from rat (female) whole brain, 10 brain regions and the pituitary gland, and show that two-dimensional gel electrophoresis (2-DGE) using precast immobilized pH (4-7) gradient (IPG) strip gels (13 cm) in the first dimension yields clean silver nitrate stained protein profiles. Though TCA/A precipitation may not be "ideal", the important choice here is the selection of an appropriate lysis buffer (LB) for solubilizing precipitated proteins. Our results reveal enrichment of protein spots by use of individual brain regions rather than whole brain, as well as the presence of differentially expressed spots in their proteomes. Thus individual brain regions provide improved protein coverage and are better suited for differential protein detection. Moreover, using a phosphoprotein-specific dye, ingel detection of phosphoproteins was demonstrated. Representative high-resolution silver nitrate stained proteome profiles of rat whole brain total soluble protein are presented. Shortcomings apart (failure to separate membrane proteins), gel-based proteomics remains a viable option, and 2-DGE is the method of choice for generating high-resolution proteome maps of rat brain and brain regions.

• 분석과학
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• 제35권1호
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• pp.8-14
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• 2022

Many studies have shown that advanced glycation end-products (AGEs) and glycation adducts are significantly linked to aging and disease. Particularly, the level of glycation in human milk is important because the AGE intake is closely related to AGE levels in infants. In this study, we used human milk samples obtained from four primiparae and four multiparae. We isolated proteins using acetone and trichloroacetic acid (TCA) precipitation. A total of 67 glycated proteins and 122 glycated peptides was quantified; among them, 19 glycated peptides were differentially expressed. We confirmed that the degree of glycation differed according to fertility. The study provides a foundation for using proteomics to evaluate the mother's milk quality and link between maternal health and human milk quality.

• 한국식품과학회지
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• 제28권1호
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• pp.99-104
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• 1996

탄수화물을 갖고 있지 않은 caseinomacropeptide (CMP)를 감성유청분말로부터 12% TCA법에 의해 분리한 결과 100 g의 감성유청분말로 부터 2.7 g을 얻을 수 있었다. 분리된 CMP는 전기영동과 아미노산 조성을 분석한 결과 매우 순도가 높은 CMP로 나타나 12% TCA침전법은 감성유청분말로 부터 CMP를 분리하는 효과적인 방법임을 알 수 있었다. 트립신을 pore glass beads에 carbodiimide (EDC) 방법으로 amide결합에 의해 고정화시켰으며 고정화된 트립신의 양은 1 g glass beads당 20 mg이었다. 수용성 트립신에 의한 CMP 가수분해는 24시간이 지나면 거의 완료되었으며 고정화 트립신은 24시간이 지난 후에도 가수분해가 진행되고 있었다. 가수분해물을 Bio-Gel P4 column에 의해 분획한 후 혈소판 응집 저해작용을 측정한 결과 고정화 트립신에 의해 24시간 가수분해시킨 CMP 분해물이 가장 높은 혈소판 응집 억제능을 보였다.

• KSBB Journal
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• 제17권3호
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• pp.261-265
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• 2002

생리활성을 가지는 펩타이드에 관한 연구는 각종 질환의 진단, 예방 및 치료약으로서의 응용 가능성이 매우 높아서 현재 많은 연구가 진행 중이다. 펩타이드를 RP-HPLC에 의해 정량, 정성분석 함으로 다양한 연구 분야로의 확대를 모색해 볼 수 있다. 혈액 속에 있는 적혈구내의 펩타이드를 분리하기 위해 0.75% TFA가 포함된 물과 아세토나이트릴을 이동상과 선형적 구배용매 조성을 이용한 실험결과, 펩타이드의 peak 분리도는 상당히 우수하였다. 혈액을 채취한 날짜의 경과에 따라서 새로운 생리활성 물질이 만들어짐을 확인할 수 있었다. 이와 같은 현상은 적혈구내의 세포들이 산소를 계속적으로 소비함으로써 산소 부족현상 때문에 발생하는 것으로 알려져 있고 구조적 변화를 나타내는 생리활성 펩타이드를 정성적으로 확인함으로써 좀더 의학적인 분야로의 연구를 수 행할 수 있게 될 것이다.