A simple procedure for removal of triton X-100 from protein samples

Holloway, Peter W.

doi:10.1016/0003-2697(73)90436-3

Cited by 839 publications

(248 citation statements)

References 7 publications

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“…By an appropriate choice of the extraction conditions, a fractionation of the intrinsic proteins could be achieved, resulting in a band 3 protein fraction of rather high purity and yield. Removal of the detergent from protein solutions by Bio Beads SM-2 is rapid and effective [31]. The presence of lipid during removal of the detergent is essential for reconstitution.…”

Section: Discussionmentioning

confidence: 99%

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Freeze‐Fracture Appearance and Disposition of Band 3 Protein from the Human Erythrocyte Membrane in Lipid Vesicles

Gerritsen

Verkley

Zwaal³

et al. 1978

European Journal of Biochemistry

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Single bilayer lipid vesicles were formed by removal of Triton X‐100 with Bio Beads SM‐2 from a mixture of egg lecithin and a Triton X‐100 extract of human erythrocyte ghosts. Upon freeze‐fracturing, these vesicles showed intramembrane particles, similar to those seen in the erythrocyte membrane. Similar particles were also observed when a partially purified band 3 preparation was used instead of the crude Triton X‐100 extract. In the reconstituted vesicles an equal distribution of the intramembrane particles between the two fracture faces was observed. This is in contrast to the unequal distribution of the particles in the erythrocyte membrane, which did not seem to be altered by removal of the extrinsic proteins. From digestion studies with trypsin and chymotrypsin of vesicles, reconstituted from the crude X‐100 extract, it is concluded that band 3 protein in the vesicle bilayer has a similar orientation as in the native membrane.

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Section: Discussionmentioning

confidence: 99%

“…In some experiments a 0.5% Triton X-100 solution in 18 mM sodium phosphate, pH 8.0, without protein was used instead of the Triton X-100 extract of membrane material. The dispersion was treated with Bio Beads SM-2 (0.3 g wet beads/ml) for 2 h at 4 "C [31].…”

Section: Preparation Of Reconstituted Vesiclesmentioning

confidence: 99%

Freeze‐Fracture Appearance and Disposition of Band 3 Protein from the Human Erythrocyte Membrane in Lipid Vesicles

Gerritsen

Verkley

Zwaal³

et al. 1978

European Journal of Biochemistry

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“…NCAM purified from newborn (P0 NCAM) or adult rats (P40 NCAM) according to published procedures [14] is detritonized using SM2 biobeads [15]. Protein is sonicated for 10 s immediately before use and is then immobilized (2/~g/ml; 1 h at 22°C) on nitrocellulose paper discs (5.5 mm diameter).…”

Section: Determination Of Affinity Constantsmentioning

confidence: 99%

Characterization of the kinetics of neural cell adhesion molecule homophilic binding

Moran

Bock

1988

FEBS Letters

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A solid-phase assay has been developed for the investigation of the kinetics of neural cell adhesion molecule (NCAM) binding. Using this assay we can show that NCAM binds to itself in a time-dependent and saturable manner. Binding constants (KB values) of 6.9 x 10 -s M and 1.23 x 10 -~ M, respectively, were obtained for adult and newborn rat NCAM homophilic binding. Binding is specifically inhibited by Fab' fragments of polyclonal anti-NCAM antibodies but is unaffected by heparin or ehondroitin sulphate. This indicates that the NCAM homophilic binding site is separate from and independent of the heparin-binding site and that a developmental modification, probably polysialation, gives rise to marked differences in the adhesive properties of NCAM.

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“…Protein concentrations were determined by the Lowry method, with bovine serum albumin as a standard. To remove the Triton X-100, which would interfere in the protein determinations, a column of Bio-beads (Bio-Rad) was used (Holloway, 1973).…”

Section: Methodsmentioning

confidence: 99%

Purification and partial characterization of the membrane-bound haem-containing proteins from Acinetobacter calcoaceticus LMD 79.41

Dokter

Wielink

Geerlof

et al. 1990

Journal of General Microbiology

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Cytoplasmic membranes of Acinetobucter cdcoaceticus cells, cultured under acetate-limiting conditions, contain cytochrome bSs4 and a cytochrome o-containing oxidase. Both have been purified to homogeneity and characterized. Cytochrome bss4 is a monomeric protein (molecular mass 70 kDa) with a midpoint potential of + lo0 mV (in the membrane it has most probably a value of +50 mV). The cytochrome o-containing oxidase seems to be an aPy8 protein since the molecular mass of the native protein was estimated to be 150 kDa and the molecular masses of the subunits, determined by SDS-polyacrylamide-gel electrophoresis, are 55, 41, 33 and 17 kDa. Redox spectroscopy of the purified complex shows the presence of a cytochrome bss5,563 having a midpoint potential of approximately + 160 mV (both in purified form and in the membrane). CO difierence spectroscopy shows the presence of a second b-type cytochrome, viz. cytochrome 0. Cytoplasmic membranes of A. calcoaceticus cells grown under oxygen-limiting conditions also show the presence of the cytochrome bSs4 and the cytochrome o-containing oxidase. In addition a protein has been solubilized with the spectral characteristics of a cytmhrome d-containing oxidase. The cytochrome o-and d-containing oxidases appear to be similar to those reported for Esckrichia coli and Profeus mirabilis. On the other hand, cytochrome bSs4 has no counterpart in these organisms since the cytochrome bSs6 described for E. cofi is quite dissimilar.

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A simple procedure for removal of triton X-100 from protein samples

Cited by 839 publications

References 7 publications

Freeze‐Fracture Appearance and Disposition of Band 3 Protein from the Human Erythrocyte Membrane in Lipid Vesicles

Freeze‐Fracture Appearance and Disposition of Band 3 Protein from the Human Erythrocyte Membrane in Lipid Vesicles

Characterization of the kinetics of neural cell adhesion molecule homophilic binding

Purification and partial characterization of the membrane-bound haem-containing proteins from Acinetobacter calcoaceticus LMD 79.41

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