Evaluation of urinary proteins by sodium dodecyl sulphate polyacrylamide disc gel electrophoresis and molecular mass analysis

Patterns of urinary proteins were examined on sodium dodecyl sulphate polyacrylamide gels and compared to those obtained using high voltage agarose electrophoresis. Both were found to be well adapted to routine laboratory analysis although the former had more sensitivity for early renal changes. Glomerular, mixed and tubular patterns were identifiable, and special precautions and pitfalls whilst interpreting the latter were pointed out. Restricted α₁ antitrypsin was shown in renal transplant proteinuria and its possible mechanisms were discussed. Molecular masses and frequencies of several discrete bands in heavy proteinurics were calculated, and attempts to identify them were made with reference to standard proteins.     

尿蛋白SDS-琼脂糖凝胶电泳方法建立及临床应用

目的　建立应用于临床的尿蛋白十二烷基硫酸钠 (SDS) 琼脂糖凝胶 (AGE)电泳方法。方法　分别对Ⅰ、Ⅱ、Ⅲ、Ⅳ型 4种琼脂糖、不同的缓冲对、电压和电泳时间进行实验 ,确定最佳的分离条件 ,建立测定方法。结果　当Ⅳ型琼脂糖凝胶浓度为4 0g/L ,SDS含量为 1g/L ,在pH 7.0的AMP 咪唑 HCl缓冲体系下进行电泳 ,可以有效地将尿蛋白按分子量大小进行分离。 5 7份临床尿蛋白阳性标本电泳结果表明 ,生理性蛋白尿 2 4例 ,中、高分子蛋白尿 5例 ,小分子蛋白尿 11例和混合型蛋白尿 17例。结论　本法具有分离效果好、操作简便、灵敏度高、成本低廉等优点 ,可以将尿蛋白按分子量大小分为小分子、中分子以及混合型 ,对肾脏疾病的受损部位和损伤程度的判断具有较高的价值 ,适于基层实验室的常规开展。     

Analysis for cerebrospinal fluid proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Cerebrospinal fluid (CSF) proteins with molecular masses of < 150,000 Da were identified by immunoblotting after two kinds of nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). With PAGE 1 (17-27% gradient gel), CSF proteins were clearly separated into seven to nine bands with molecular masses of 3000-67,000 Da; seven bands were identified as beta 2-microglobulin, lysozyme, prealbumin, free kappa and lambda chain, apolipoprotein A-I, glycoproteins, and albumin by immunoblotting. With PAGE 2 (10-20% gradient gel), proteins were clearly separated into 11-16 bands with molecular masses of 15,000-150,000 Da; 11 were identified as prealbumin, free kappa and lambda chain, apolipoprotein A-I, glycoproteins, albumin, alpha 1-antitrypsin, transferrin (separated into two bands), immunoglobulin fragments, haptoglobin, and IgG. We analyzed CSF samples collected from 81 patients with cerebrospinal signs by these SDS-PAGE methods and observed prominent bands in some cases.     

A simple, sensitive technique for classification of apolipoprotein(a) isoforms by sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Lipoprotein(a) (Lp(a)) is a lipoprotein containing a unique glycoprotein, apolipoprotein(a) (apo(a)), which shows considerable heterogeneity of apparent molecular mass on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). A unifying classification of isoform has been lacking. A simple sensitive procedure for classifying apo(a) isoforms was developed in which the relative mobility of apo(a) on SDS-PAGE was related to that of apolipoprotein (apo) B-100 (Rf vs B). After Western blotting apo(a) bands were visualised by a sensitive double antibody technique employing commercial polyclonal antibodies (sheep antihuman Lp(a) antibody, alkaline phosphatase-linked donkey antisheep antibody). The technique was sensitive (lower limit of detection 0.02 micrograms apo(a)) and had good reproducibility (coefficient of variation 0.9-6.4%). Ten isoform mobilities are described (less than 0.35, 0.40, 0.50, 0.60, 0.70, 0.80, 1.0, 1.10, greater than 1.15). Individuals may have single or double band phenotypes. This classification is compatible with those previously described and the method is suitable for many laboratories, as it employs standard equipment and commercially available materials.     

Synthesis of polymers of Bence-Jones protein for use as molecular weight markers for sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

A simple method is described of preparing a series of stable polymers of Bence-Jones protein, of known molecular weight, for use as molecular weight markers for sodium dodecyl sulphate (SDS) - polyacrylamide gel electrophoresis.     

SDS-AGE尿蛋白电泳在糖尿病肾病早期诊断中的应用

目的探讨SDS-AGE非浓缩尿蛋白电泳在糖尿病肾病早期诊断中的作用。方法对20例对照组及50例糖尿病患者的尿液进行尿常规蛋白定性,然后在法国Sebia公司HYDRASYS全自动电泳仪上,采用十二烷基硫酸钠-琼脂糖凝胶进行电泳(SDS-AGE)。同时利用BN-100特种蛋白测定仪检测尿微量白蛋白。结果尿蛋白经SDS-AGE电泳后,根据蛋白质的分子量的大小顺序排列在凝胶上。对照组电泳后只有2例出现单纯淡染的白蛋白条带。糖尿病组有14例无蛋白条带出现、15例出现单纯白蛋白带、9例出现小分子蛋白带、5例出现大分子蛋白带、7例出现混合性蛋白带。其中有2例尿常规蛋白定性结果分别为±和 ,但电泳条带结果显示为混合性蛋白带;在15例出现单纯白蛋白带的病例中,有6例在尿常规蛋白定性为阴性。结论SDS-AGE非浓缩尿蛋白电泳,操作简便,无须浓缩尿液,省时,尿蛋白按分子量大小予以分离,结果清晰便于保存。能正确的反映尿液内各蛋白组分的含量及变化,对糖尿病肾病的早期诊断、鉴别诊断、指导治疗具有较大价值。     

SDS-AGE尿蛋白电泳在糖尿病肾病早期诊断中的应用

目的 探讨SDS-AGE非浓缩尿蛋白电泳在糖尿病肾病早期诊断中的作用.方法 对20例对照组及50例糖尿病患者的尿液进行尿常规蛋白定性,然后在法国Sebia公司HYDRASYS全自动电泳仪上,采用十二烷基硫酸钠-琼脂糖凝胶进行电泳(SDS-AGE).同时利用BN-100特种蛋白测定仪检测尿微量白蛋白.结果 尿蛋白经SDS-AGE电泳后,根据蛋白质的分子量的大小顺序排列在凝胶上.对照组电泳后只有2例出现单纯淡染的白蛋白条带.糖尿病组有14例无蛋白条带出现、15例出现单纯白蛋白带、9例出现小分子蛋白带、5例出现大分子蛋白带、7例出现混合性蛋白带.其中有2例尿常规蛋白定性结果分别为±和+.但电泳条带结果显示为混合性蛋白带;在15例出现单纯自蛋白带的病例中,有6例在尿常规蛋白定性为阴性.结论 SDS-AGE非浓缩尿蛋白电泳,操作简便,无须浓缩尿液,省时,尿蛋白按分子量大小予以分离.结果清晰便于保存.能正确的反映尿液内各蛋白组分的含量及变化,对糖尿病肾病的早期诊断、鉴别诊断、指导治疗具有较大价值.     

Rapid differentiation of glomerular and tubular proteinuria by sodium dodecyl sulfate polyacrylamide gel electrophoresis

Serum and urine protein samples were incubated with sodium dodecyl sulfate and subjected to polyacrylamide gel electrophoresis. There was a linear relationship between the logarithm of the molecular weight of the protein and its distance of migration (R_F) on the gel. This relationship held over a molecular weight range from at least 160000 to 12000, i.e., over a range particularly useful for serum proteins and urine proteins. This system was then applied to the analysis of both serum and urine proteins. Glomerular proteinuria (characterized by the presence of albumin and plasma proteins of a molecular size larger than albumin) and tubular proteinuria (characterized by the presence of albumin and proteins of molecular size smaller than albumin) were readily distinguished. The results were similar to those obtained on the same samples with Sephadex G-200 and Sephadex G-75 columns. The SDS polyacrylamide gel electrophoretic method resolved the proteins of varying molecular weights better than did Sephadex. It was simple to use, and very much more rapid; thus, it was suitable for the examination of large numbers of samples.     

Analysis of proteinuria in health and disease using sodium dodecyl sulphate-acrylamide gel electrophoresis

Abstract. Sodium dodecyl sulphate—acrylamide gel electrophoresis (SDS-AGE) separates proteins according to their molecular weight (MW) without being influenced by their electrostatic charge. It was carried out on the urine of 17 healthy control subjects and 92 ambulatory patients with suspected or known renal impairment. The SDS-AGE patterns were classified as physiological, low MW predominance, middle MW predominance, high MW predominance, or mixed low and high MW. Patients were separately classified as having either normal kidneys or glomerular, tubular or mixed renal lesions according to the results of clinical investigation. Comparison of both classifications revealed that SDS-AGE allowed a correct diagnosis of the site of renal involvement in 83 per cent of cases. Predominantly low MW protein excretion correlated with tubular damage. Middle and high MW patterns correlated with glomerular disease, and a relationship between nephrotic syndrome with minimal glomerular changes and middle MW pattern was found in two cases, suggesting that SDS-AGE could be used to evaluate the selectivity of proteinuria. However, the estimation of selectivity by SDS-AGE should be studied in a sufficient number of cases and compared with measurements of selectivity by the protein clearance method.     

Reliable identification of clinically prevalent species and subspecies of staphylococci by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis

We identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 10 species and 5 subspecies of Staphylococcus among 139 clinical isolates and compared it with conventional tests. All isolates showed species-specific whole-cell protein profiles, even atypical strains, with up to 60% and at least 73.5% of interspecies and intraspecies similarity, respectively. Except for 5 isolates that presented biochemical profiles of Staphylococcus hominis subsp. hominis, but were identified as S. hominis subsp. novobiosepticus by SDS-PAGE, there was 100% accordance between the methods used. Comparison with the partial 16S-rDNA sequences confirmed by SDS-PAGE showed the high accuracy of this method to identify staphylococci subspecies and species.     

Simplified sodium dodecyl sulfate-polyacrylamide gel electrophoresis of unconcentrated urine with enhanced resolution and detection sensitivity

We applied a simple sodium dodecyl sulfate-polyacrylamide gel electrophoresis method to urine. The method, developed for serum protein analysis (Clin Chem 1984;30:475-9), has a high sample throughput and gives excellent resolution with unconcentrated urine. It clearly distinguishes and characterizes proteinuric urine (7.5 microL) by Coomassie Blue staining and gives complex silver-stained patterns with nonproteinuric urine (2 microL). The former is recommended for routine clinical screening, the latter for research purposes.     

Analysis of the glycoprotein and protein composition of Bernard-Soulier platelets by single and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Previous reports have described conflicting results concerning the glycoprotein (GP) and protein composition of Bernard-Soulier platelets. In view of this controversy we have analyzed the platelets of four Bernard-Soulier patients using improved single and two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis procedures. An absence of staining for carbohydrate of membrane GP Ib was characteristic for the platelets of each patient. Major periodate-Schiff staining bands corresponding to membrane GP IIb, IIIa, and IIIb were clearly detected and their presence was confirmed by two-dimensional SDS-polyacrylamide gel electrophoresis. The protein content of the Bernard-Soulier platelets was increased two- to fourfold. However, analysis of their protein composition using 7-12% acrylamide gradient gels showed normal polypeptide profiles. Lactoperoxidase-catalyzed 125I-labeling of the Bernard-Soulier platelet surface proteins was followed by SDS-polyacrylamide gel electrophoresis and autoradiography. No labeling in the Ib position was detected whereas the other major membrane GP, including Ia and IIa, were normally located. In contrast, GP Ib was clearly detected by periodate-Schiff staining and autoradiography when normal human platelets that had been exhaustively treated with neuraminidase before the lactoperoxidase-catalyzed iodination were analysed. No abnormalities were detected in the GP patterns of membranes isolated from the patients' erythrocytes. Only a severe molecular abnormality or possible deletion of GP Ib could account for this major platelet lesion in the Bernard-Soulier syndrome.