A simple method for dialysis of small-volume samples

Marusyk, R. G.; Sergeant, Alain

doi:10.1016/0003-2697(80)90477-7

Cited by 175 publications

(76 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Refolding was allowed to continue for 30 min. Microdialysis for refolding was performed by the method of Marusky and Sergeant (17). Typically, 85 l of the solution containing denatured GroEL was placed on a VMWP Millipore membrane (0.05 m), which was made to float on 20 ml of the dialysate in a Petri dish that was then covered to permit equilibration.…”

Section: Methodsmentioning

confidence: 99%

Refolding and Reassembly of Active Chaperonin GroEL After Denaturation

Ybarra

Horowitz

1995

Journal of Biological Chemistry

View full text Add to dashboard Cite

Conditions are reported that, for the first time, permit the folding and assembly of active chaperonin, GroEL, following denaturation in 8 M urea. The folding could be achieved by dilution or dialysis, and the best yields required the simultaneous presence of ammonium sulfate and the Mg 2؉ complexes of ATP or ADP. Ammonium sulfate was the key to this particular protocol, since there was a small recovery of oligomer in its presence, but no detectable recovery was induced by ATP or ADP without ammonium sulfate. The refolded/reassembled GroEL could arrest the spontaneous folding of rhodanese, and it could participate in the chaperonin-assisted refolding of rhodanese as effectively as GroEL that had never been unfolded. The results demonstrate that the primary sequence of GroEL contains the information required for its folding, assembly, and function. Thus, in contrast to previous studies, although chaperonins may facilitate GroEL folding, they are not necessary for the acquisition of the functional oligomeric state of this chaperone. This ability to fold denatured GroEL in vitro will facilitate studies of the influences that determine the interesting folding pattern adopted by the native protein.

show abstract

Section: Methodsmentioning

confidence: 99%

Refolding and Reassembly of Active Chaperonin GroEL After Denaturation

Ybarra

Horowitz

1995

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…topoisomerase I (Promega) or with rat liver topoisomerase I (gift from G. Muzard) for 30 min at 370C. After two phenol extractions, the relaxed form of DNA (F-Il) was ethanol precipitated (14), resuspended in 20 Al of 10 mM Tris HCl/0.4 mM EDTA, pH 7.6, microdialyzed using a Millipore VSWP 2500 membrane (15) (2). The mixture was incubated at 37°C for 10 min, and the reaction was terminated by the addition of 5 ml of cold 2x SSC (0.3 M NaCl/0.03 M trisodium citrate).…”

mentioning

confidence: 99%

The noncovalent complex between DNA and the bifunctional intercalator ditercalinium is a substrate for the UvrABC endonuclease of Escherichia coli.

Lambert

Jones²,

Roques³

et al. 1989

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

View full text Add to dashboard Cite

We have demonstrated that the noncovalent complex formed between DNA and an antitumor bifunctional intercalator, ditercalinium, is recognized in vitro as bulky covalent DNA lesions by the purified Escherichia coli UvrABC endonuclease. It was established that no covalent drug-DNA adduct was formed during the incubation ofthe drug with DNA or during subsequent incubation with the UvrAB proteins. The nucleoprotein-ditercalinium complexes appear different from those generated by repair of pyrimidine dimers. The UvrA protein is able to form a stable complex with ditercaliniumintercalated DNA in the presence of ATP, whereas both UvrA and UvrB proteins are required to form a stable complex with pyrimidine dimer-containing DNA. The apparent half-life of the UvrA-and UvrAB-ditercalinium-DNA complexes following removal of free ditercalinium is 5 min. However, if the free ditercalinium concentration is maintained to allow the intercalation of one molecule of ditercalinium per 3000 base pairs, the half-life of the UvrA-or UvrAB-ditercalinium-DNA complex is 50 min, comparable to that of the complex of UvrAB proteins formed with pyrimidine dimer-containing DNA.UvrABC endonuclease incises ditercalinium-intercalated DNA as efficiently as pyrimidine dimer-containing DNA. However, unlike repair of pyrimidine diners, the incision reaction is strongly favored by the supercoiling of the DNA substrate. Because UvrA-or UvrAB-ditercalinium-DNA complexes can be formed with relaxed DNA without leading to a subsequent incision reaction, these apparently dead-end nucleoprotein complexes may become lesions in themselves resulting in the cytotoxicity of ditercalinium. Our results show that binding of excision repair proteins to a noncovalent DNA-ligand complex may lead to cell toxicity.

show abstract

“…6) In both intramolecular and intermolecular ligation tests, we obtained approximately 3-5 times more transformants when we desalted by drop dialysis, as compared with the more commonly used silica-based microcolumn purification. In this study, we tested a single brand of microcolumn (the EZNA MicroElute Cycle Pure kit from Omega Bio-Tek).…”

mentioning

confidence: 89%

“…5) However, an older study showed that drop dialysis, in which a 5-100 mL drop is placed on a floating membrane filter, can also result in effective desalting, with DNA recovery rates of 98-99%. 6) Here we present a rigorous comparison of the two desalting methods, and show that drop dialysis is preferred in the construction of large libraries.…”

mentioning

confidence: 99%