One-step purification of mouse monoclonal antibodies from ascitic fluid by DEAE Affi-gel blue chromatography

Bruck, Claudine; Portetelle, Daniel; Glineur, Cécile; Bollen, Alex

doi:10.1016/0022-1759(82)90178-8

Cited by 303 publications

(82 citation statements)

References 5 publications

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“…For SSV immunoisolation purposes, M48 monoclonal antibodies against Syt I (50) were purified with DEAE blue resin (Bio-Rad) (51) and then dialyzed extensively against distilled water. The purified IgG fraction (0.4 mg) was coupled to Eupergit C1Z methacrylate microbeads (98.6 mg; 1-m diameter; Röhm Pharma, Darmstadt), previously washed with distilled water, by incubation at room temperature for 1 h (52).…”

Section: Methodsmentioning

confidence: 99%

Calcium-dependent Oligomerization of Synaptotagmins I and II

Osborne

Herreros

Bastiaens

et al. 1999

Journal of Biological Chemistry

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Synaptotagmins constitute a large family of membrane proteins characterized by their distinct distributions and different biochemical features. Genetic evidence suggests that members of this protein family are likely to function as calcium sensors in calcium-regulated events in neurons, although the precise molecular mechanism remains ill defined. Here we demonstrate that different synaptotagmin isoforms (Syt I, II, and IV) are present in the same synaptic vesicle population from rat brain cortex. In addition, Syt I and II co-localize on the same small synaptic vesicle (SSV), and they heterodimerize in the presence of calcium with a concentration dependence resembling that of the starting phase of SSV exocytosis (EC 50 ‫؍‬ 6 ؎ 4 M). The association between Syt I and Syt II was demonstrated by immunoprecipitation of the native proteins and the recombinant cytoplasmic domains and by using fluorescence resonance energy transfer (FRET). Although a subpopulation of SSV containing Syt I and IV can be isolated, these two isoforms do not show a calcium-dependent interaction. These results suggest that the self-association of synaptotagmins with different calcium binding features may create a variety of calcium sensors characterized by distinct calcium sensitivities. This combinatorial hypothesis predicts that the probability of a single SSV exocytic event is determined, in addition to the gating properties of the presynaptic calcium channels, by the repertoire and relative abundance of distinct synaptotagmin isoforms present on the SSV surface.Neuronal communication depends upon the transduction of an electric nerve impulse for the release of neurotransmitters from their storage compartment, the small synaptic vesicles (SSV).1 This tightly regulated process is triggered by the rapid increase of the intracellular calcium due to the opening of voltage-gated calcium channels at the active zones of the synaptic plasma membrane, which causes the activation of the SSV fusion machinery (1, 2). Of the proteins involved in the physiological cycle of the SSV, the members of the synaptotagmin family represent the best candidates for the role of calcium sensors in neurotransmitter release and, more generally, in regulated exocytosis (3-5). This protein family comprises more than a dozen members having the same overall structure and a variable degree of homology (4, 5). Synaptotagmins are integral membrane proteins with broad distributions in neuronal and non-neuronal tissues that vary between isoforms (4, 5). In the central nervous system and neuroendocrine cells, synaptotagmins are localized on SSV and secretory granules (6). These proteins are characterized by a single membrane-spanning domain and by a large cytoplasmic portion containing two internal repeats that have homology with the C2 domain of protein kinase C (7,8). This domain is known to regulate the calciumdependent translocation of protein kinase C to membranes and is present in many proteins with different functions (5, 8). In synaptotagmins, the two C2 domains are re...

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Section: Methodsmentioning

confidence: 99%

Calcium-dependent Oligomerization of Synaptotagmins I and II

Osborne

Herreros

Bastiaens

et al. 1999

Journal of Biological Chemistry

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“…Splenocytes from immune spleens and Ag8.653 myeloma cells were mixed at a cell ratio of 5: 1 and fused in 1.5ml of 45% polyethylene glycol 4000 (Merck, Darmstadt, FRG) Radiobinding assays HEA 125 was purified from ascites fluid by DEAE Affi-Gel Blue (Biorad, Munic, FRG) chromatography (Bruck et al, 1982). Radioiodination of MAb was performed by a slight modification of the chloramine-T method (Greenwood et al, 1963) Figure 1 illustrates the strong membrane staining of fixed ME-180 cervix carcinoma cells by HEA125.…”

Section: Methodsmentioning

confidence: 99%

Epithelium-specific surface glycoprotein of Mr 34,000 is a widely distributed human carcinoma marker

et al. 1987

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“…B. Xin (Institute of Microbiology and Epidemiology, Beijing 100850, China). It contained mAb 2G8 raised against AChE from electric organ of T. nacline timilei [9] which was purified according to Wang et al [13] by protein-A-Sepharose CL-4B chromatography (Pharmacia Fine Chemicals, Uppsala, Sweden) or purified by chromatography on DEAE -Affi-gel blue (Bio-Rad) according to Bruck et al [37]. mAb 4F19, raised against human erythrocyte AChE, was obtained as published previously [S].…”

Section: Antibodiesmentioning

confidence: 99%

The monoclonal antibody 2G8 is carbohydrate‐specific and distinguishes between different forms of vertebrate cholinesterases

Liao

Heider

Sun³

et al. 1991

European Journal of Biochemistry

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The monoclonal antibody (mAb) 2G8 (subclass IgG2,) raised against acetylcholinesterase (AChE, EC 3.1.1.7) from electric organ of Torpedo nacline timilei crossreacted with AChE from Torpedo marmorata, electric eel (Electrophorus electricus), flounder (Platichthys Jlesus) body muscle, rat brain, bovine brain, and human brain, this suggests that the epitope to which mAb 2G8 bound had been highly conserved during evolution. No crossreaction was found with AChE from human and bovine erythrocytes, nor with butyrylcholinesterase (BtChE, EC 3.1.1 3) from human serum. Binding of mAb 2G8 to the globular G z form of AChE from T. marmorata strongly decreased enzyme activity, while no significant inhibition was found with either collagen-tailed, asymmetric forms, or with the enzymes from flounder body muscle or mammalian sources. The possibility that mAb 2G8 bound to anionic sites of AChE could be excluded since neither edrophonium chloride nor decamethonium bromide influenced the binding of 2G8 to the enzymes. Enzyme-linked immunosorbent assay and Western blot showed that heat-denatured, diisopropylfluorophosphate-treated, CNBr-and trypsin-digested AChE from T. marmorata still reacted with mAb 2G8; this indicates that the epitope to which 2G8 bound, at least partially, belonged to a continuous determinant. Treatment of cholinesterases with N-glycosidase F abolished crossreaction with 2G8, showing that an essential part of the epitope consisted of N-linked carbohydrates.

show abstract

One-step purification of mouse monoclonal antibodies from ascitic fluid by DEAE Affi-gel blue chromatography

Cited by 303 publications

References 5 publications

Calcium-dependent Oligomerization of Synaptotagmins I and II

Calcium-dependent Oligomerization of Synaptotagmins I and II

Epithelium-specific surface glycoprotein of Mr 34,000 is a widely distributed human carcinoma marker

The monoclonal antibody 2G8 is carbohydrate‐specific and distinguishes between different forms of vertebrate cholinesterases

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