Glanzmann's thrombasthenia is a rare autosomal recessive hemorrhagic disorder characterized by prolonged bleeding time, ad deficient or absent clot retraction in the presence of normal platelet count. The major underlying abnormality in this disease is grossly defective first-phase aggregation of platelet, which are unresponsive to ADP or other platelet agonists such as epinephrine, collagen, thrombin in any concentration. This disability is caused by a decrease or absence of the platelet membrans glycoprotein IIb-IIIa complex, a member of the integrin family of adhesive receptors involved in cell-cell and cell-matrix fibronectin, and vitronectin On the development of surface labeling technique, a variety of biochemical techniques such as radioimmunoassay, crossed immunoelectrophoresis and SDS-PAGE have been used to study the structure and the function of platelet membrane glycoproteins, and to detect the platelet functional defect. But all of these techniques demand a relatively large amount of homogeneous paletelet population that requires manipulation through isolation and washing procedures before analysis. In order to eliminaste such an intricate procedure, we have applied method for analyzing platelet surface components in whole blood using monoclonal antibody and flow cytometry to recognize the absence of severe reduction of platelet membrane glycoprotien llb-llla complex. Platelet analysis by flow cytometry is a successful alternative rapid diagnostic technique for Glanzmann's thrombasthenia patients as well as well as for carriers of this disease. Fow cytometry technique provides a sensitive tool for investigating platelet functional defects caused by altered expression or deficiency of platelet surface proteins.
Adenosine Diphosphate ; Adhesives ; Bleeding Time ; Blood Platelets* ; Clot Retraction ; Collagen ; Electrophoresis, Polyacrylamide Gel ; Epinephrine ; Fibronectins ; Flow Cytometry* ; Glycoproteins ; Hemorrhagic Disorders ; Humans ; Immunoelectrophoresis, Two-Dimensional ; Membrane Glycoproteins* ; Membrane Proteins ; Membranes* ; Platelet Count ; Platelet Membrane Glycoproteins ; Radioimmunoassay ; Thrombasthenia* ; Thrombin ; Vitronectin

BACKGROUND AND OBJECTIVE: Buckwheat is considered one of the most important food allergens in Korea. Although a very small amount is ingested or inhaled, it can cause serious allergic reactions. However, the major allergens of buckwheat still remain to be elucidated. The aim of our study was to identify and characterize the major allergen of buckwheat seed. MATERIAL AND METHOD: Dialysis membrane with a cut-off MW 1kD was used for the preparation of crude buckwheat seed allergen extract. SDS-PAGE under reducing conditions and IgE immunoblotting were performed using sera from 15 buckwheat sensitive subjects. Isoelectric focusing and lectin blotting assay were done. RESULT: Western blot analysis showed more than 15 IgE-reactive buckwheat proteins. Among them, a 24kD protein was shown to be the most frequently bound to sera from allergic subjects (54%). Isoelectric point of 24kD protein was around 5.9. In lectin blotting assay, 24kD protein did not bind to Con A nor five other lectins. CONCLUSION: A 24kD protein was the most frequently recognized allergenic component in buckwheat seed. Isoelectric point was around 5.9. Glycosylation was not detected in 24kD of buckwheat protein.
Allergens ; Blotting, Western ; Dialysis ; Electrophoresis, Polyacrylamide Gel ; Fagopyrum* ; Glycosylation ; Hypersensitivity ; Immunoblotting ; Immunoglobulin E ; Isoelectric Focusing ; Isoelectric Point ; Korea ; Lectins ; Membranes

Adult ; Blood Glucose ; analysis ; Diabetes Mellitus ; blood ; Electrophoresis, Agar Gel ; Electrophoresis, Paper ; Female ; Hemoglobin A ; analysis ; Humans ; Male

OBJECTIVESTo analyze human spermatozoa membrane proteins by two-dimensional gel electrophoresis and to provide a basis for drawing the protein map of normal human spermatozoa membrane proteins.

METHODSSpermatozoa were purified by Percoll density centrifugation, and spermatozoa membrane proteins were analyzed by two-dimensional gel electrophoresis using isoelectric focusing and polyacrylamide gel electrophoresis.

RESULTSAbout 800 protein spots could be identified by the imaging analysis system. The molecular weight and isoelectric point of most proteins were 20,000 to 100,000 and 3.0 to 7.0 respectively.

CONCLUSIONSThere are more than 800 types of proteins in the human spermatozoa membrane. The spermatozoa membrane protein can be identified with a certain precision by two-dimensional gel electrophoresis.

Electrophoresis, Gel, Two-Dimensional ; Humans ; Image Processing, Computer-Assisted ; Isoelectric Focusing ; Isoelectric Point ; Male ; Membrane Proteins ; chemistry ; Molecular Weight ; Spermatozoa ; chemistry

Mugwort and ragweed pollens have been considered as important respiratory allergens in Korea. These two pollens are abundant in the air of Seoul from August through October. Many ragweed-sensitive patients have shown concurrent sensitivities to mugwort pollen. However the antigenic relationship between these two pollens has not been clarified. To observe the cross-reactivity between them, we developed polyclonal anti-mugwort and anti-ragweed antibodies by immunization on New Zealand white rabbits, and performed crossed immunoelectrophoresis(CIE) with two pollen extracts. Five precipitation lines were formed by mugwort and anti-mugwort antibody. One precipitation line was formed by ragweed and anti-ragweed antibody. There was no reaction from mugwort and anti-ragweed antibody, and from ragweed and anti-mugwort antibody. These results indicate that there is no cross-antigenicity between mugwort and ragweed pollens.
Animal ; Antibodies/immunology ; Cross Reactions ; Immunoelectrophoresis, Two-Dimensional ; Pollen/*immunology ; Rabbits

OBJECTIVETo evaluate the effects of two sample preparation methods on two-dimensional polyacrylamide gel electrophoresis (2-DE)-based serum protein separation, and produce high-resolution and reproducible 2-DE images for identifying disease-related serum protein.

METHODSDirect solubilization and hot SDS methods were used separately to extract and handle the total proteins of serum samples from patients with colorectal carcinoma. Immobilized pH gradient 2-DE was used to separate the total proteins. After image analysis of silver-stained 2-D gels, 3 differential protein spots were identified by matrix-assisted laser desorption/time-of-flight mass spectrometry.

RESULTSThe total proteins treated with hot SDS method were used to perform 2-DE. 2-DE patterns with high resolution and reproducibility were obtained for human serum samples. 2-DE was performed 3 times for the samples treated by direct solubilization and hot SDS methods, respectively, resulting in the average number of spots of 675-/+46 and 702-/+49, respectively. The average matching protein spots were 573-/+42 and 623-/+52, with average matching rate of 85.3% and 89.6%, respectively. The average position deviation of matched spots in different gels was 0.85-/+0.30 mm and 0.81-/+0.28 mm in IEF direction, and 1.02-/+0.18 mm and 0.97-/+0.12 mm in SDS-PAGE direction. Mass spectrometry of the 2-D gels treated with hot SDS method generated high-quality mass spectra, and the sample preparation method allowed detection of relatively low abundance protein.

CONCLUSIONHot SDS method is more effective for human serum protein sample preparation and well-resolved, reproducible 2-DE profiles of human serum have been established in this study.

Blood Protein Electrophoresis ; Blood Proteins ; analysis ; isolation & purification ; Electrophoresis, Gel, Two-Dimensional ; methods ; Electrophoresis, Polyacrylamide Gel ; methods ; Humans ; Peptide Mapping ; Protein Interaction Mapping ; Reproducibility of Results

BACKGROUND AND OBJECTIVES: Calretinin is a neuron specific, high affinity cytosolic calcium-binding protein of the EF-hand family. In the mammalian inner ear, it is expressed in the organ of Corti and most of the spiral ganglion neurons. Authors observed the change of expression and amount of calretinin according to the maturation in the rat cochleae. MATERIALS AND METHOD: Sprague-Dawley rat cochleae collected from each stage (postnatal day (P) P5, P17, P35) were analyzed using 2D gel electrophoresis, proteomic analysis, Western blot analysis, and fluorescence immunocytochemistry. RESULTS: In P17 and P35, calretinin was identified at an isoelectric point (pI) of 4.9 and a molecular weight of 29 kDa in the analysis of the rat cochlea proteins using proteomic analysis. P17 and P35 revealed remarkable existence of calretinin in 2D gel electrophoresis and the Western blot, whereas P5 demonstrated no existence in 2D gel electrophoresis and weak expression in the Western blot. In fluorescence immunocytochemistry, P17 and P35 showed intense calretinin immunoreactivity in the inner hair cells and most ganglion neurons, but P5 displayed no immunoreactivity in inner hair cells and very weak expression in spiral ganglion cells. CONCLUSION: Compared with the early neonatal stage, an amount of calretinin remarkably increases during the critical period of the cochlear maturation and is maintained until the young adult stage. These results suggest that calretinin may have a specific role as a calcium-binding protein since the cochlear maturation in the rat inner ear.
Animals ; Blotting, Western ; Calbindin 2* ; Cochlea* ; Critical Period (Psychology) ; Cytosol ; Ear, Inner ; Electrophoresis, Gel, Two-Dimensional ; Fluorescence ; Ganglion Cysts ; Hair ; Humans ; Immunohistochemistry ; Isoelectric Point ; Molecular Weight ; Neurons ; Organ of Corti ; Rats* ; Rats, Sprague-Dawley ; Spiral Ganglion ; Young Adult

OBJECTIVETo analyse clinical feasibility of two electrophoresis procedures of seminal plasma proteins, agarose gel electrophoresis and SDS-agarose gel electrophoresis.

METHODSSixty-nine semen samples were examined and classified into three groups: the asthenozoospermia (n = 22), the asthenoteratozoospermia (n = 19), and the relative normal group (n = 28) with normal routine and special test results, according to WHO routine and special test criterion. Then, the seminal plasma protenis were separated by two different electrophoresis, with SDS-agarose and agarose support medium, the buffer pH 7.0 and 9.2 respectively. The agarose gel electrophoresis was done under various sample loading time, motion power and staining modules. The completed gels were scanned and compared the each other statistically.

RESULTSSeminal plasma proteins can be separated into 4 strips by SDS-agarose gel electrophoresis with acid crystal violet, and the strips were diffusion and with dark background. However, 6 clear strips named A, B, C, D, E, and F can be obtained by agarose gel electrophoresis with 6 min. After samples were loaded and stained by amidoblack, there showed appropriate spaces among strips, and it was very easy to scan the drying gel by a densitometer. Using agarose gel electrophoresis, the statistical difference in strip C and E was shown between the asthenozoospermia and the relative normal group, and between the asthenozoospermia and the asthenoteratozoospermia, however, not between the relative normal and the asthenoteratozoospermia group. Moreover, the samples in the relative normal group with normal routine and special test results were in different electrophoresis maps.

CONCLUSIONAgarose gel electrophoresis of seminal plasma proteins with buffer pH 9.2, 6 min. sample loading and amidoblack stain was a simple, fast and fit technique for clinic.

Adult ; Electrophoresis, Agar Gel ; methods ; Electrophoresis, Polyacrylamide Gel ; methods ; Humans ; Male ; Proteins ; analysis ; Semen ; chemistry ; Staining and Labeling

OBJECTIVETo explore the feasibility of membrane protein-based two-dimensional electrophoresis (2-DE) in the investigations of chondrocyte-related diseases and its complementarity with total protein-based 2-DE.

METHODSKnee cartilage samples were obtained to isolate the chondrocytes with type II collagenase/hyaluronidase digestion. The membrane proteins and total proteins were extracted and loaded separately onto PH3.0-10.0 non-linear gel strip for 2-DE analysis. The qualities of membrane protein-based 2-DE gels were evaluated, and the difference between the distribution profiles of the membrane protein spots and that of the total protein were observed and their complementarities were evaluated.

RESULTSMembrane protein-based 2-DE generated high-quality gel images, and on each gel 412.3±13.5 protein spots were identified. These spots were distributed in the region of isoelectric point pH 5.0-9.0. In the gel images generated by the total proteins, 564.3±5.9 protein spots were identified in each image, and the spots were distributed in the region of isoelectric point pH 3.0-7.0.

CONCLUSIONMembrane protein-based 2-DE of the chondrocytes can generate high-quality gel images, and the isoelectric distribution of the protein spots is complementary to that of total protein, which provides valuable information for chondrocyte-related diseases.

Adult ; Chondrocytes ; chemistry ; Electrophoresis, Gel, Two-Dimensional ; methods ; Female ; Humans ; Isoelectric Point ; Male ; Membrane Proteins ; isolation & purification ; Middle Aged

Lactate dehydrogenase was purified 21-fold from liver of Varanus bengalensis using colchicine-sepharose column chromatography. The crude enzyme showed two isoenzymes (LDH-5 and LDH-4) by agarose gel electrophoresis (AGE). The purified enzyme showed a single band after SDS-PAGE corresponding to molecular mass of 35 kDa. The molecular mass of native enzyme was about 140 kDa. The optimum pH for the forward reaction was 7.5 while that for the reverse reaction was pH 9.5. The K-m values for pyruvate, NADH, lactate and NAD(+) were 0.17 +/- 0.037, 0.02 +/- 0.004, 12.4 +/- 3.05 and 0.38 +/- 0.032 mM, respectively. Pre-heating of enzyme showed that its t(50) was 40-50 degrees C. Oxalate and n-hexanediol were inhibitors for both forward and reverse reactions. Among divalent ions, Cu++ was shown to be more effective inhibitor for the forward reaction.
Chromatography ; Electrophoresis, Agar Gel ; Electrophoresis, Polyacrylamide Gel ; Hydrogen-Ion Concentration ; Ions ; Isoenzymes ; L-Lactate Dehydrogenase* ; Lactic Acid* ; Liver* ; NAD ; Pyruvic Acid