• Preventive Nutrition and Food Science
• /
• v.7 no.1
• /
• pp.43-47
• /
• 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
• /
• v.11 no.3
• /
• pp.215-222
• /
• 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 the Korean Society of Food Science and Nutrition
• /
• v.27 no.6
• /
• pp.1117-1124
• /
• 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.

• Journal of Applied Biological Chemistry
• /
• v.51 no.3
• /
• pp.88-94
• /
• 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.

• Applied Biological Chemistry
• /
• v.39 no.2
• /
• pp.123-126
• /
• 1996

Simple and rapid method of purification of plasma proteins from bloods of slaughtered animals was developed and the proteins were applied to plywood products as a blood 히ue to utilize waste materials. Plasma protein was obtained by adding 2% trichloroacetic acid (TCA) or 0.6 N HCI as optimal concentration to the supernatant, after centrifugation of bloods. Molecular properties of beef and pig plasma proteins were examined on SDS-PAGE. Application of blood glue to plywood was quite satisfactory compared to the synthetic amino resin by tensile-shear test for the strength of adhesive bonding.

• Korean Journal of Food Science and Technology
• /
• v.28 no.1
• /
• pp.99-104
• /
• 1996

Carbohydrate-free caseinomacropeptide (CMP) was isolated from the sweet whey powder by a precipitation method using 12% trichloroacetic acid. The yield of carbohydrate-free CMP was 2.7 g from 100 g sweet whey powder. The electrophoretic pattern and the amino acid analysis of CMP showed that isolated CMP was quite pure, indicating the precipitation with 12% trichloroacetic acid was very effective for isolating carbohydrate-free CMP from the sweet whey powder. Trypsin, covalently immobilized on pore glass beads by carbodiimide (EDC) method, was 20mg per 1g glass beads. CMP was almost completely hydrolyzed by soluble trypsin in 24hr, but not by immobilized trypsin. The tryptic hydrolysates were fractionated on a Bio-Gel P 4 column $(1.5{\times}120\;cm)$and separated peptides were tested for their capacities to inhibit platelet aggregation using a aggregometer. The hydrolysate obtained from CMP after 24hr digestion by immobilized trypsin showed the highest activity.

• Analytical Science and Technology
• /
• v.35 no.1
• /
• pp.8-14
• /
• 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.

• Molecules and Cells
• /
• v.22 no.1
• /
• pp.119-125
• /
• 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.

• KSBB Journal
• /
• v.17 no.3
• /
• pp.261-265
• /
• 2002

The biologically active peptides in human blood can adjust the functions of many physiological systems. The peptides in human blood were separated on the five steps of linear gradient-elution mode by RP-HPLC with UV detection. The size of commercially available $C_{18}$ chromatographic column was 4.60$\times$150 mm with particle size of 5 $\mu\textrm{m}$ and pore size of 100 $\AA$. The mobile phases used were water in 0.75% trifluoroacetic acids (TFA) and organic modifier of acetonitrile. The isolation methods suggested in this work for peptides from the blood were composed of the formation of immiscible liquid layers and precipitation by centrifuge and chemicals of sodium citrate and trichloroacetic acid (TCA). The some peptides were identified based on the retention times previously constructed database.

• The Korean Journal of Nuclear Medicine
• /
• v.28 no.1
• /
• pp.106-111
• /
• 1994

2-iminothiolane is known to bind $NH_2$ group of lysine in the protein and deliver SH group, which can be used to label protein with $^{99m}Tc$. In this study, we looked for the best reaction condition in which 2-iminothiolane is conjugated to human polyclonal IgG and labeling condition with $^{99m}Tc$-glucoheptonate. Labeling yield was measured with TSK G4000SW column and HPLC or precipitation with 10% TCA (trichloroacetic acid) and 1% HSA. In vivo distribution was investigated with Staphylococcal abscess bearing rats. With decreasing glucoheptonate, the labeling yield decreased. Without 2-iminothiolane, $^{99m}Tc$-glucoheptonate was bound to IgG, which seemed to be direct labeling. With increasing 2-iminothiolane upto 20 times higher than IgG, the labeling yield increased, and plateau was seen with higher molar excess of 2-iminothiolane. Polymer formation was not observed. The pH for the conjugation of 2-iminothiolane and IgG was best around 6.4. $^{99m}Tc$-2-iminothiolane-IgG showed faster blood clearance, higher renal activity and lower hepatic and splenic activity than $^{99m}Tc$-DTPA-IgG. The biodistribution of $^{99m}Tc$-2-iminothiolane-IgG with higher molar excess of 2-iminothiolane was not different from that with lower molar excess. Labeling antibodies with $^{99m}Tc$ using 2-iminothiolane can afford a possible route to simple labeling and wide clinical use of the immunoscintigraphy.