New detection method after cellulose acetate membrane isoelectric focusing: Activity staining for lactate dehydrogenase, detection for sugar chains using lectin and simple Western blotting

Iijima, Shiro; Kimura, Miyako; Shiba, Kiyoko

doi:10.1002/(sici)1098-2825(1999)13:5<234::aid-jcla8>3.0.co;2-x

Cited by 6 publications

(2 citation statements)

References 16 publications

(16 reference statements)

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“…Immunostaining of proteins on cellulose acetate membranes usually involves acid fixation using sulfosalicylic acid or trichloroacetic acid 28,29 . Acid can hydrolyse sialic acid, an important terminal sugar of the glycoprotein carbohydrate chains.…”

Section: Discussionmentioning

confidence: 99%

A fixation method for the optimisation of western blotting

Sun

Huang

et al. 2019

Sci Rep

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Western blotting is the most extensively used technique for the identification and characterisation of proteins and their expression levels. One of the major issues with this technique is the loss of proteins from the blotted membrane during the incubation and washing steps, which affects its sensitivity and reproducibility. Here, we have optimised the fixation conditions for immunoblotting and lectin blotting on electroblotted polyvinylidene difluoride and nitrocellulose membranes, using a combination of organic solvents and heating. Loss of proteins from polyvinylidene difluoride membranes was greatly reduced using this approach, the intensity of lectin blotting and immunoblotting was shown to increase 2.8- to 15-fold and 1.8- to 16-fold, respectively, compared with that samples without treated. Using the optimised method, cystic fibrosis transmembrane regulator and hypoxia-inducible factor 1, two difficult-to-analyse proteins with important physiological and pathological roles, were effectively detected. Additionally, it may help the identification of novel diagnostic markers for prostate cancer.

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Section: Discussionmentioning

confidence: 99%

A fixation method for the optimisation of western blotting

Sun

Huang

et al. 2019

Sci Rep

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“…Immunostaining of proteins on cellulose acetate membranes usually involves fixation via acid treatment with sulfosalicylic acid or trichloroacetic acid . Immunostaining of SMME membranes, however, does not require acid fixation.…”

Section: Resultsmentioning

confidence: 99%

Serum protein fractionation using supported molecular matrix electrophoresis

2013

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Supported molecular matrix electrophoresis (SMME), in which a hydrophilic polymer such as PVA serves as a support within a porous PVDF membrane, was recently developed. This method is similar to cellulose acetate membrane electrophoresis but differs in the compatibility to glycan analysis of the separated bands. In this report, we describe the first instance of the application of SMME to human serum fractionation, and demonstrate the differences with serum fractionation by cellulose acetate membrane electrophoresis. The SMME membrane exhibited almost no EOF during electrophoresis, unlike the cellulose acetate membrane, but afforded comparative results for serum fractionation. The visualization of each fraction was achieved by conventional staining with dye such as Direct Blue-71, and objective quantification was obtained by densitometry after inducing membrane transparency with 1-nonene. Immunostaining was also achieved. Moreover, mass spectrometric analysis of both N-linked and O-linked glycans from the separated bands was demonstrated. Serum fractionation and glycan profiling of each fraction using SMME will enable novel insights into the relationships between various glycosylation profiles and disease states.

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Improved method for immunostaining of mucin separated by supported molecular matrix electrophoresis by optimizing the matrix composition and fixation procedure

et al. 2011

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Mucins are a family of heavily glycosylated high molecular mass proteins that have great potential as novel clinical biomarkers for the diagnosis of various malignant tumors. Supported molecular matrix electrophoresis (SMME) is a new type of membrane electrophoresis that can be used to characterize mucins. In SMME, mucins migrate in a molecular matrix supported by membrane materials. Here, we have developed an immunostaining method for the identification of SMME-separated mucins. The novel method involves stably fixing the mucins onto the SMME membrane and optimizing the molecular matrix for the fixation process. We applied this technique for the detection of MUC1 produced from three cancer cell lines (T47D, HPAF-II and BxPC3) and also analyzed their O-linked glycans by mass spectrometry. Our results revealed that properties of the MUC1 molecules from the three cell lines are different in terms of migrating position in SMME and glycan profile. The present method allows simple and rapid characterization of mucins in terms of both glycans and core proteins. The method will be a useful tool for the exploration of mucin alterations associated with various diseases such as cancer.

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New detection method after cellulose acetate membrane isoelectric focusing: Activity staining for lactate dehydrogenase, detection for sugar chains using lectin and simple Western blotting

Cited by 6 publications

References 16 publications

A fixation method for the optimisation of western blotting

A fixation method for the optimisation of western blotting

Serum protein fractionation using supported molecular matrix electrophoresis

Improved method for immunostaining of mucin separated by supported molecular matrix electrophoresis by optimizing the matrix composition and fixation procedure

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