Chapter 26 Affinity Chromatography

Urh, Marjeta; Simpson, Dan; Zhao, Kate

doi:10.1016/s0076-6879(09)63026-3

Cited by 108 publications

(50 citation statements)

References 33 publications

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“…Researchers have used the concept of affinity in a variety of applications, ranging from chemical and protein purification to enzyme synthesis to drug targeting. [9,10] However, application as a mechanism to control the rate of release is relatively new. In this paper, various affinitybased drug delivery systems will be examined in detail, as well as a discussion of implications and future directions in this exciting field.…”

Section: Introductionmentioning

confidence: 99%

Affinity‐Based Drug Delivery

Wang

Recum

2010

Macromolecular Bioscience

175

146

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Affinity-based drug delivery systems utilize interactions between the therapeutic drug and the delivery system to manipulate drug loading and to control drug release. In this paper, affinity-based drug delivery system syntheses, types of therapeutic factors delivered, and delivery system loading and release are discussed in detail. The paper is divided into three subsections, based on the type of delivery system: molecular imprinting systems, growth-factor delivery, and cyclodextrin-based delivery. The objective of this paper is to examine the current state of research, highlight the breakthroughs and challenges, point out potential impacts of this relatively new technology, and explore future developmental areas.

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Section: Introductionmentioning

confidence: 99%

Affinity‐Based Drug Delivery

Wang

Recum

2010

Macromolecular Bioscience

175

146

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“…Two interacting molecules form the cognate groups of an affinity capture system (1,2). One group is the protein of interest or an affinity tag appended to the protein of interest via genetic engineering, resulting in expression of a tagged fusion protein within a model organism.…”

Section: Affinity Capture: Principlesmentioning

confidence: 99%

“…Batch binding promotes thorough mixing of the affinity medium with the clarified cell extract, maximizing binding in minimal time. We also use spherical, non-porous, micron-scale, paramagnetic beads, rather than traditional porous resins; because the affinity binding occurs on the bead surface, there are no size exclusion limitations that can bias the capture of large complexes assembled with the protein of interest, as can occur with traditional chromatographic media (2,106,133,134). We were unable to release bound LINE-1 RNPs from anti-FLAG-agarose beads using native elution by competitive displacement with 3xFLAG peptide (and comparable outcomes have been seen by us and others with protease cleavage and Sepharose) (135), although the complexes were readily released from magnetic anti-FLAG medium by this approach (25).…”

Section: Affinity Capture: Practicementioning

confidence: 99%

Affinity Proteomics to Study Endogenous Protein Complexes: Pointers, Pitfalls, Preferences and Perspectives

et al. 2015

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Dissecting and studying cellular systems requires the ability to specifically isolate distinct proteins along with the co-assembled constituents of their associated complexes. Affinity capture techniques leverage high affinity, high specificity reagents to target and capture proteins of interest along with specifically associated proteins from cell extracts. Affinity capture coupled to mass spectrometry (MS)-based proteomic analyses has enabled the isolation and characterization of a wide range of endogenous protein complexes. Here, we outline effective procedures for the affinity capture of protein complexes, highlighting best practices and common pitfalls.

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“…In this regard many of the biochemistry or physical interaction studies being carried out are associated with unique challenges presented by large-scale screening and the immobilization to solid substrates that in some cases may generate significant non-specific binding and high levels of background in the assays performed. The HaloTag technology offers some discrete and potentially important advantages to address these two issues based on the covalent and very high affinity interaction between the HaloTag and its ligand [15, 16, 32]. The covalent linkage of the HaloTag to immobilized surfaces ensures that high stringency washes may be performed without concern of removing the immobilized proteins [33].…”

Section: Potential Advantages Of Covalent Linkagementioning

confidence: 99%

The HaloTag: Improving Soluble Expression and Applications in Protein Functional Analysis

Peterson

Kwon²

2013

CCG

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Technological and methodological advances have been critical for the rapidly evolving field of proteomics. The development of fusion tag systems is essential for purification and analysis of recombinant proteins. The HaloTag is a 34 KDa monomeric protein derived from a bacterial haloalkane dehalogenase. The majority of fusion tags in use today utilize a reversible binding interaction with a specific ligand. The HaloTag system is unique in that it forms a covalent linkage to its chloroalkane ligand. This linkage permits attachment of the HaloTag to a variety of functional reporters, which can be used to label and immobilize recombinant proteins. The success rate for HaloTag expression of soluble proteins is very high and comparable to maltose binding protein (MBP) tag. Furthermore, cleavage of the HaloTag does not result in protein insolubility that often is observed with the MBP tag. In the present report, we describe applications of the HaloTag system in our ongoing investigation of protein-protein interactions of the Y. pestis Type 3 secretion system on a custom protein microarray. We also describe the utilization of affinity purification/mass spectroscopy (AP/MS) to evaluate the utility of the Halo Tag system to characterize DNA binding activity and protein specificity.

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Chapter 26 Affinity Chromatography

Cited by 108 publications

References 33 publications

Affinity‐Based Drug Delivery

Affinity‐Based Drug Delivery

Affinity Proteomics to Study Endogenous Protein Complexes: Pointers, Pitfalls, Preferences and Perspectives

The HaloTag: Improving Soluble Expression and Applications in Protein Functional Analysis

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