We describe an improved Phos-tag SDS-PAGE (Zn²⁺-Phos-tag SDS-PAGE) using a dizinc(II) complex of Phos-tag acrylamide in conjunction with a neutral-pH gel system buffered with Bis-Tris hydrochloride (Bis-Tris-HCl) to detect shifts in the mobility of phosphorylated proteins. Our alternative technique (Mn²⁺-Phos-tag SDS-PAGE) using a polyacrylamide-bound Mn²⁺-Phos-tag and a conventional Laemmli's buffer system under alkaline pH conditions has limitations for separating certain phosphoproteins. The major improvements and utilities were demonstrated by visualizing novel up-shifted bands of commercially available pepsin, recombinant substrate protein tau treated in vitro with tyrosine kinases, and endogeneous β-catenin in whole cell lysates. Additionally, the Zn²⁺-Phos-tag SDS-PAGE gels showed better long-term stability than the Mn²⁺-Phos-tag SDS-PAGE gels. We can thus present a simple, convenient, and more reliable "in-house" gel system for phosphate-affinity SDS-PAGE.

抄録全体を表示

The presence of 2-mercaptoethanol or dithiothreitol in the protein denaturing solution used to prepare samples for SDS-PAGE gives rise to artifacts by silver staining of the gel. Addition of iodoacetamide to the reduced protein sample prevents the appearance of artifacts. Silver staining is strengthened by the dithiothreitol treatment but weakened by subsequent treatment with iodoacetamide. High concentration of dithiothreitol also inhibited development of silver staining. These effects of iodoacetamide or high concentration of dithiothreitol were observed more pronouncedly with the silver-staining method of Merril et al.⁴⁾ than of Guevara.⁵⁾ Mobility of the protein having intramolecular disulfide bonds is more or less slowed down depending on the concentration of dithiothreitol used. From these results it is concluded that the most suitable concentration of dithiothreitol is in the range of 40 to 80mM from the viewpoints of sensitivity of silver staining, completion of mobility shift, and sharpness of developed protein bands.

抄録全体を表示

Abnormal IgA molecule, which lacked in disulfide bridges between α chains, was detected from the sera of patients of monoclonal IgA gammopathy with albuminuria by the method of urea-SDS-PAGE performed in the reducing-agent-free condition. Throughout the procedure, protein in the specimen was kept away from any reducing agent to avoid an artificial production of half molecules during the experiment. A band of 80kDa protein, which showed immunoreactivity to both anti-α- and anti-λ-chain antibodies, was detected in sera from four patients of monoclonal IgA gammopathy accompanied with albuminuria. The molecular mass and the immunoreactivity indicated that the 80kDa protein was so-called“IgA half molecule”(α₁λ₁). The band of α₁λ₁ molecule was faint on the gel in the specimens from patients of no albuminuria. The abnormal α₁λ₁ molecule was eluted from the column of gel permeation chromatography into fractions corresponding to 320kDa when it was performed using urea-free buffer. The chromatographic behavior suggested the non-covalent interaction between the abnormal α₁λ₁ molecules in the non-denaturing buffer condition.

抄録全体を表示

Phosphorylation is a major post-translational modification widely used in regulation of many cellular processes. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase activated by activation subunit p35. Cdk5-p35 regulates various neuronal activities such as neuronal migration, synaptic activity, and cell death. The kinase activity of Cdk5 is regulated by proteolysis of p35: proteasomal degradation causes downregulation of Cdk5 whereas cleavage of p35 by calpain causes overactivation of Cdk5. Phosphorylation of p35 determines the proteolytic pathway. We have previously identified Ser8 and Thr138 as major phosphorylation sites using metabolic labeling of cultured cells followed by 2D-phosphopeptide mapping and phospho-specific antibodies. However, these approaches cannot determine the extent of p35 phosphorylation in vivo. Here we report the use of Phos-tag SDS-PAGE to reveal the in vivo phosphorylation states of p35. Using Phos-tag acrylamide, electrophoretic mobility of phosphorylated p35 was delayed because it is trapped at Phos-tag sites. We constructed phosphorylation-dependent banding profiles of p35 and Ala substitution mutants at phosphorylation sites co-expressed with Cdk5 in COS-7 cells. Using the standard banding profiles, we assigned respective bands of endogenous p35 with combinations of phosphorylation states, and quantified Ser8, Ser91 and Thr138 phosphorylation. This is the first quantitative and site-specific measurements of phosphorylation of p35, demonstrating the usefulness of Phos-tag SDS-PAGE for analysis of phosphorylation states of in vivo proteins.

抄録全体を表示

Serum ultrafiltrate was obtained from hemodialysis patients by using an extra corporeal ultrafiltration method equipped with a hollow fiber dialyser, and electrophoresed on the SDS-polyacrylamide gel having a high resolving power in the low molecular weight region. Among the major bands less than 40KD observed with plasma of hemodialysis patient, the bands of 29K, 28K, 25K, 22K, 16.7K and 8.9K molecular weights were also found in serum ultrafiltrate of the same patient. By an immunological method, the following bands were identified: α₁-microglobulin (28K), retinol-binding protein (22K), prealbumin (16.7K) and β₂-microglobulin (8.9K). As compared with serum ultrafiltrate obtained from healthy person, 28K, 25K (in some cases), 22K and 8.9K bands showed obvious increase in cases of hemodialysis patients except for one patient who showed an urine volume of 200ml/day and an electrophoretic pattern closed to that of healthy person.

抄録全体を表示

SDS-電気泳動と immuno-blot 法を用いて, I型およびII型コラーゲンの混在試料中のII型コラーゲンの定量法について検討した.
蛋白染色法では, 多量のI型コラーゲンを含むII型コラーゲン混在試料を用いた場合, 全α₁鎖の染色性が理論値よりも低下するという問題を有するものの, I型コラーゲンが0.24μg以下, II型コラーゲンが0.51μg以下においては10%以内の誤差で混在試料中のII型コラーゲンの定量が可能であった.
一方, immuno-blot 法では, 1.5ngのII型コラーゲンを検出でき, II型コラーゲンのみ含む試料を用いた場合, 3ngから75ngの範囲内で高い定量性を示した. また, 240ngのI型コラーゲンを含む混在試料を用いた場合でも, 免疫染色時間を短縮することにより, 600ng以下のII型コラーゲンを特異的に定量できた.

抄録全体を表示

In order to identify urinary proteins excreted from normal subjects, we employed a new method by which molecular weight and immunochemical property of urinary proteins can be determined simultaneously. Urine was subjected to linear gradient (3∼40%) SDS-PAGE and then transferred to nitrocellulose membrane by electrophoretic blotting method. The membrane was stained with Auro Dye and blotted proteins were identified by enzyme immunoassay using specific antibodies. In this method, we identified more than 12 protein bands from urine, that is, α₂-macroglobulin, tubular epithelial antigen, Tamm-Horsfall glycoprotein, IgA, IgG, transferrin, albumin, α₁-microglobulin, L-chain (dimer, monomer), retinol-binding protein, and β₂-microglobulin. In addition, we found native forms and some fragments of immunoglobulins. In this report, we analyzed only normal subjects.
These results suggest that this method is useful for analysis not only of glomerular or tubular proteins but also tissue antigens from urine with various kinds of renal diseases.

抄録全体を表示