Electrophoretic transfer of proteins across polyacrylamide membranes

Rylatt, Dennis B.; Napoli, Mark; Ogle, David; Gilbert, Anna C.; Lim, Sian Yik; Nair, C H

doi:10.1016/s0021-9673(99)00807-9

Cited by 45 publications

(26 citation statements)

References 8 publications

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“…Membranes designed to restrict the movement of any proteins from stream 1 and stream 2 were prepared as described previously [9]. Cartridges consisting of the separation membrane sandwiched between two grids and two restriction membranes were assembled as described previously for use in the Gradiflow BF200 unit [6].…”

Section: Membrane Preparationmentioning

confidence: 99%

“…The cooling system of recirculating low conductivity buffer ensures the material is not denatured by heat during the separating process. Separation across the membrane is affected by numerous parameters as described previously [6].…”

Section: Preparative Membrane Electrophoresismentioning

confidence: 99%

“…The latter are useful for the recovery of biomaterials including proteins and DNA with the potential for the removal of other entities including viruses from biological samples [2][3][4][5]. Commercially available separation media for preparative-scale electrophoresis systems such as Gradiflow  are usually prepared by the copolymerization of acrylamide (AAm) with N,N'-methylenebisacrylamide (Bis) [6].…”

Section: Introductionmentioning

confidence: 99%

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Poly(vinyl alcohol) hydrogels: Their synthesis and steps towards control of electroendosmosis

2003

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Poly(vinyl alcohol) (PVAl) hydrogel networks cross-linked with glutaraldehyde were prepared and their properties as membranes examined using a variety of techniques including preparative electrophoresis. Electroendosmosis (EEO) was observed and shown to be the result of charges on the membrane and of complexation with borate buffer ions. Investigation of "glutaraldehyde" solutions showed acid entities in, or formed in "glutaraldehyde" were responsible for EEO. Techniques for using "glutaraldehyde" which minimize EEO are described.

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Section: Membrane Preparationmentioning

confidence: 99%

Section: Preparative Membrane Electrophoresismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Poly(vinyl alcohol) hydrogels: Their synthesis and steps towards control of electroendosmosis

2003

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“…The Gradiflow concept was first outlined by Margolis et al [6]. Purification is based on scalable preparative electrophoresis [7], and the principle that proteins have an inherent charge determined by their amino acid composition and the pH of the surrounding milieu. Plasma solution is introduced into the sample stream and pumped tangentially past the separation membrane where a voltage placed across the plasma facilitates the movement of charged molecules.…”

Section: Introductionmentioning

confidence: 99%

Blood protein purification and simultaneous removal of nonenveloped viruses using tangential-flow preparative electrophoresis

Evtushenko

Wang²,

Stokes

et al. 2005

Electrophoresis

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Gradiflow is new technology allowing purification of important blood proteins from viral contaminated plasma. Protein purification is based on unique scalable tangential-flow preparative electrophoresis, and is distinct from current technology because protein purification and virus removal are performed in the same step. This one-step removal and purification exploits both the size and charge of target proteins. The medically important blood proteins, immunoglobulin G (IgG) and alpha-1-antitrypsin, were chosen to demonstrate the ability of this process to purify proteins from contaminated plasma. Clearance factors achieved by infectivity assays and polymerase chain reaction (PCR) that meet regulatory requirements demonstrated removal of canine parvovirus (CPV). CPV is a model virus for pathogenic nonenveloped viruses, including parvovirus B19, not adequately removed or inactivated by most processes currently in practice. The recovery of proteins from plasma with high purity, recovery, and function, while simultaneously removing viruses, provides blood products with a level of purity compatible with clinical use more quickly and cheaply than available techniques.

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“…The system uses thin polyacrylamide membranes and operates on different principles compared to the devices based on isoelectric membranes [11,12]. Membranes are either restrictive by pore size (size-based separations) or nonrestrictive (charged-based separations).…”

Section: Introductionmentioning

confidence: 99%

Impact of prefractionation using Gradiflow™ on two‐dimensional gel electrophoresis and protein identification by matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry

Pang¹,

Fryksdale²,

Chow³

et al. 2003

Electrophoresis

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Prefractionation of protein samples prior to two-dimensional electrophoresis (2-DE) has the potential to increase the dynamic detection range for proteomic analysis. We evaluated a membrane-based electrophoretic separation technique (Gradiflow) for its ability to fractionate an exoproteome sample from the filamentous fungus Trichoderma reesei. The sample was separated on the basis of size and charge. Buffer optimization was found to be necessary for successful size fractionation. Fractionation by charge was used to resolve the sample into four fractions that were subjected to analysis by two-dimensional electrophoresis (2-DE). Enhanced detection of low-abundance proteins with selective removal of high-abundance species was achieved. Fractionated and unfractionated samples were examined for differences in the ability to identify proteins following 2-DE using trypsin in-gel digestion followed by peptide mass fingerprinting using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Fractionated samples showed marked improvement in protein identification ability and sequence coverage. This study demonstrates the utility of the Gradiflow for fractionation, resulting in an enhancement of resolution and characterization of a moderately complex proteome.

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Electrophoretic transfer of proteins across polyacrylamide membranes

Cited by 45 publications

References 8 publications

Poly(vinyl alcohol) hydrogels: Their synthesis and steps towards control of electroendosmosis

Poly(vinyl alcohol) hydrogels: Their synthesis and steps towards control of electroendosmosis

Blood protein purification and simultaneous removal of nonenveloped viruses using tangential-flow preparative electrophoresis

Impact of prefractionation using Gradiflow™ on two‐dimensional gel electrophoresis and protein identification by matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry

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