Semiquantitative analysis of urinary low protein levels using silver dot blot assay

Matsuda, Kazuyuki; Hiratsuka, Nobuo; Kurihara, Yoshitaka; Shiba, Kiyoko

doi:10.1002/jcla.1022

Cited by 17 publications

(16 citation statements)

References 8 publications

(5 reference statements)

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“…In this study, a rapid and highly sensitive colloidal silver staining reagent suitable for use with cellulose acetate membranes, as reported by Matsuda et al (1,2) and Hiratsuka et al (3), was used. First, 15 urinary proteins in samples from patients with tubulointerstitial nephritis were identified, fractionated, and analyzed.…”

Section: Introductionmentioning

confidence: 99%

Cellulose acetate membrane electrophoresis in the analysis of urinary proteins in patients with tubulointerstitial nephritis

Kubota¹,

Machii²,

Hiratsuka³

et al. 2003

Clinical Laboratory Analysis

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Urinary proteins from 14 patients with tubulointerstitial nephritis were analyzed by cellulose acetate membrane electrophoresis. Urinary total protein concentrations were measured, and urinary 15 proteins (prealbumin, albumin, alpha(1)-microglobulin, alpha(1)-antitrypsin, alpha(2)-macroglobulin, haptoglobin, retinol binding protein, transferrin, beta(2)-microglobulin, IgA, IgG, kappa- and lambda-light chains, cystatin C, and lysozyme) were identified by the use of a rapid and highly sensitive colloidal silver staining reagent suited for use with cellulose acetate membranes, as reported previously by Matsuda et al. (J Clin Lab Anal 15:171-174, 2001; Clin Chem47:763-766, 2001) and Hiratsuka et al. (J Clin Lab Anal 10:403-406, 1996). We also analyzed urinary total protein concentration and urinary protein fractions according to the presence of acute or nonacute interstitial nephritis. In addition, the relationship between urinary protein fraction and complications of interstitial nephritis was analyzed. The goal of this work was to find a useful index for the diagnosis of tubulointerstitial nephritis.

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Section: Introductionmentioning

confidence: 99%

Cellulose acetate membrane electrophoresis in the analysis of urinary proteins in patients with tubulointerstitial nephritis

Kubota¹,

Machii²,

Hiratsuka³

et al. 2003

Clinical Laboratory Analysis

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“…The protein concentration in urine samples was measured by the use of the calibrator solution (Fractorol, Jokoh, Tokyo, Japan) blotted along with the samples onto the CA membrane, followed by silver colloidal staining as described previously 8 …”

Section: Methodsmentioning

confidence: 99%

Rapid and sensitive electrophoresis of urinary protein clearly reveals the pathophysiological feature of renal diseases

Sakatsume

Kubota²,

Ogawa

et al. 2007

Nephrology

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“…The final pellet was suspended in phosphatebuffered saline (PBS) or commercial RIPA lysis buffer (Pierce, Rockford, IL, USA) containing 25 mmol/L tris (hydroxymethyl) aminomethane, 150 mmol/L NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS (pH 7.6) 12,13) , and then incubated for 1 hour at room temperature. The total protein concentrations of the final pellets containing the exosomes were measured using the dot blot assay with colloidal silver staining 14) .…”

Section: Exosome Purificationmentioning

confidence: 99%

Comparison of urinary exosomal protein solubilization methods for two-dimensional gel electrophoresis

et al. 2012

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SUMMARYBackground: Urinary exosomal proteins have recently emerged as important candidates for elucidating the mechanisms underlying physiological events and disease-related metabolism in the kidney. Here, we evaluated standard sample preparation methods for two-dimensional gel electrophoresis (2DE) to determine which one yielded the maximum protein recovery from urinary exosomes for protein identification.Materials and Methods: Urinary exosomes were purified from a healthy subject by using ultracentrifugation. The final pellets were dissolved with PBS or RIPA buffer. After being desalted, these exosomal protein solutions were each treated with 1 of 4 rehydration buffers (Rbs) containing detergents in the following formulations: CHAPS (Rb1), CHAPS and Triton X-100 (Rb2), dodecyl maltoside (Rb3), and ASB-14 (Rb4).Results: For all Rbs, a much greater number of protein spots was detected in the samples isolated with RIPA than with PBS. Only minor differences were observed in the number of protein spots for Rb1-3. The largest protein spots were detected using the combination of RIPA buffer and Rb4; however, the background on the 2DE gel was high in the region of >66 kD and at the lower pH values. For all combinations, the co-precipitation of the urinary Tamm-Horsfall protein masked the protein spots in the 66-100 kD region.Conclusion: For extracting a large number of proteins with a relatively clear background on silver-stained 2DE gels, the optimal exosomal protein-dissolving buffer is RIPA buffer. All of the evaluated Rbs, except for the one containing ASB-14 as a detergent, is suitable for solubilizing exosomal proteins on 2DE.

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Semiquantitative analysis of urinary low protein levels using silver dot blot assay

Cited by 17 publications

References 8 publications

Cellulose acetate membrane electrophoresis in the analysis of urinary proteins in patients with tubulointerstitial nephritis

Cellulose acetate membrane electrophoresis in the analysis of urinary proteins in patients with tubulointerstitial nephritis

Rapid and sensitive electrophoresis of urinary protein clearly reveals the pathophysiological feature of renal diseases

Comparison of urinary exosomal protein solubilization methods for two-dimensional gel electrophoresis

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