Western Blot Analysis of Epidermal Growth Factor Using Gelatin-Coated Polyvinylidene Difluoride Membranes

Nishi, Nozomu; Inui, Masashi; Miyanaka, Hiroshi; Oya, Haruyo; Wada, Fumio

doi:10.1006/abio.1995.1302

Cited by 10 publications

(3 citation statements)

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“…Immunolocalization was performed on 5-m sections by means of the streptoavidin-biotin-alkaline phosphatase complex technique using a Histofine SAB-AP kit (Nichirei, Tokyo). Affinitypurified anti-rat EGF antibodies (14), anti-human bFGF antibodies (R & D Systems) and anti-BrdU mAbs (Progen, FIG. 1.…”

Section: Methodsmentioning

confidence: 99%

Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain

Nishi

Matsushita

Yuube

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

104

112

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The autocrine͞paracrine peptide signaling molecules such as growth factors have many promising biologic activities for clinical applications. However, one cannot expect specific therapeutic effects of the factors administered by ordinary drug delivery systems as they have limited target specificity and short half-lives in vivo. To overcome the difficulties in using growth factors as therapeutic agents, we have produced fusion proteins consisting of growth factor moieties and a collagen-binding domain (CBD) derived from Clostridium histolyticum collagenase. The fusion proteins carrying the epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) at the N terminal of CBD (CBEGF͞ CBFGF) tightly bound to insoluble collagen and stimulated the growth of BALB͞c 3T3 fibroblasts as much as the unfused counterparts. CBEGF, when injected subcutaneously into nude mice, remained at the sites of injection for up to 10 days, whereas EGF was not detectable 24 h after injection. Although CBEGF did not exert a growth-promoting effect in vivo, CBFGF, but not bFGF, strongly stimulated the DNA synthesis in stromal cells at 5 days and 7 days after injection. These results indicate that CBD may be used as an anchoring unit to produce fusion proteins nondiffusible and long-lasting in vivo.Currently, more than 100 soluble peptide signaling molecules, including peptide hormones, growth factors, and lymphokines, are known. These molecules can be classified into two types: one, like classic hormones, is produced in a specific organ͞cell and exerts a characteristic effect on relatively limited target cells via blood flow (endocrine), and the other is produced in a wide variety of tissues and acts in an autocrine or a paracrine manner with low target specificity. In the latter case, the specificity of the action depends for the most part on spatiotemporarily controlled production of the signaling molecule and the local tissue architecture. The former type of molecules (endocrine factors) are suitable as therapeutic agents as they can be delivered systemically without loss of target specificity. In contrast, autocrine͞paracrine factors may induce diverse responses in various tissues when present in the blood at concentrations higher than a threshold value. Thus, one cannot expect specific therapeutic effects of the factors administered by ordinary methods, though they have many promising biologic activities for clinical applications.

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Section: Methodsmentioning

confidence: 99%

Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain

Nishi

Matsushita

Yuube

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

104

112

View full text Add to dashboard Cite

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“…However, it is difficult to detect low-molecular-weight peptides efficiently, in part because they so readily detach from the blotted membranes. To avoid this detection problem, previous researchers have fixed the blotted membranes with aldehyde before applying them to the conventional Western blotting method with shaking 1 – 3 . Nishi et al succeeded in detecting a low-molecular-weight (6 kDa) epidermal growth factor by fixation of a gelatin-coated PVDF membrane with formaldehyde.…”

mentioning

confidence: 99%

A new approach to detect small peptides clearly and sensitively by Western blotting using a vacuum-assisted detection method

et al. 2013

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Western blotting is a widely used technique for the detection and quantification of proteins and peptides. However, it is challenging to detect small peptides efficiently by the conventional Western blotting method with shaking, in part because the peptides readily detach from the blotted membrane. Although some modified Western blotting protocols have been developed to overcome this problem, it remains difficult to prevent peptide detachment from the membrane. In this study, we show that the previously developed vacuum-assisted detection method greatly improves the detection of small peptides without additional protocol modification. The vacuum-assisted method was developed to shorten the time required for all immunodetection steps, and all the Western blotting solutions penetrated the membrane quickly and efficiently by this method. By using this vacuum method, we succeeded in detecting small peptides that were completely undetectable by the conventional Western blotting method. We also confirmed that peptide detachment was induced even by gentle shaking in the case of the conventional method, and the detachment was accelerated when detergent was present in the buffer. Unlike in the conventional method, there is no need to shake the membrane in solution in the vacuum method. Therefore, it is thought that the small peptides could be detected sensitively only by the vacuum method.

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“…Samples were subjected to SDS-PAGE on 10% (for detection of CD43 and CD45) or 12.6% (CD2, CD3 and CD7) gels. The separated proteins were transferred to polyvinylidene difluoride membranes (Millipore Co., Billerica, MA), followed by immunodetection with monoclonal antibodies against human CD2 (MT210), CD3 (UCH-T1), CD7 (H-126), CD43 (DF-T1) (Santa Cruz Biotechnology, Santa Cruz, CA), and human CD45 (Chemicon International Inc., Temecula, CA), and phosphataselabeled goat anti-mouse IgG antibodies (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD), as described previously (Nishi et al 1995).…”

Section: Western Blot Analysismentioning

confidence: 99%

Functional and structural bases of a cysteine-less mutant as a long-lasting substitute for galectin-1

et al. 2008

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Galectin-1 (Gal-1), a member of the beta-galactoside-binding animal lectin family, has a wide range of biological activities, which makes it an attractive target for medical applications. Unlike other galectins, Gal-1 is susceptible to oxidation at cysteine residues, which is troublesome for in vitro/vivo studies. To overcome this problem, we prepared a cysteine-less mutant of Gal-1 (CSGal-1) by substituting all cysteine residues with serine residues. In the case of wild-type Gal-1, the formation of covalent dimers/oligomers was evident after 10 days of storage in the absence of a reducing agent with a concomitant decrease in hemagglutination activity, while CSGal-1 did not form multimers and retained full hemagglutination activity after 400 days of storage. Frontal affinity chromatography showed that the sugar-binding specificity and affinity of Gal-1 for model glycans were barely affected by the mutagenesis. Gal-1 is known to induce cell signaling leading to an increase in the intracytoplasmic calcium concentration and to cell death. CSGal-1 is also capable of inducing calcium flux and growth inhibition in Jurkat cells, which are comparable to or more potent than those induced by Gal-1. The X-ray structure of the CSGal-1/lactose complex has been determined. The structure of CSGal-1 is almost identical to that of wild-type human Gal-1, showing that the amino acid substitutions do not affect the overall structure or carbohydrate-binding site structure of the protein. These results indicate that CSGal-1 can serve as a stable substitute for Gal-1.

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Western Blot Analysis of Epidermal Growth Factor Using Gelatin-Coated Polyvinylidene Difluoride Membranes

Cited by 10 publications

References 0 publications

Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain

Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain

A new approach to detect small peptides clearly and sensitively by Western blotting using a vacuum-assisted detection method

Functional and structural bases of a cysteine-less mutant as a long-lasting substitute for galectin-1

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