Chapter 28 Identification, Production, and Use of Polyol-Responsive Monoclonal Antibodies for Immunoaffinity Chromatography

Thompson, Nancy E.; Foley, Katherine; Stalder, Elizabeth S.; Burgess, Richard R.

doi:10.1016/s0076-6879(09)63028-7

Cited by 14 publications

(11 citation statements)

References 27 publications

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“…In order to demonstrate the ability of IFAST to isolate clinically relevant, transient protein complexes, we next focused on the biologically important, canonical Wnt signaling pathway. This pathway is involved in embryonic development and adult tissue homeostasis, and its misregulation has been linked to tumor formation in various tissues, including intestinal and breast cancer [18]. Using IFAST, we have isolated endogenous levels of the low-density lipoprotein receptor (LDLR)-related protein (LRP5) and LRP6, one of the functional interactors of LRP5 [17].…”

Section: Resultsmentioning

confidence: 99%

Weak protein–protein interactions revealed by immiscible filtration assisted by surface tension

Berry

Chin

Jackson

et al. 2014

Analytical Biochemistry

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Biological mechanisms are often mediated by transient interactions between multiple proteins. The isolation of intact protein complexes is essential to understanding biochemical processes and an important prerequisite for identifying new drug targets and biomarkers. However, low-affinity interactions are often difficult to detect. Here, we use a newly described method called immiscible filtration assisted by surface tension (IFAST) to isolate proteins under defined binding conditions. This method, that gives a near-instantaneous isolation, enables significantly higher recovery of transient complexes as compared to current wash-based protocols, which require re-equilibration at each of several wash steps, resulting in protein loss. The method moves proteins, or protein complexes, captured on a solid phase through one or more immiscible phase barriers that efficiently exclude the passage of non-specific material in a single operation. We use a previously described polyol-responsive monoclonal antibody (PR-mAb) to investigate the potential of this new method to study protein-binding. In addition, difficult-to-isolate complexes involving the biologically and clinically important Wnt signaling pathway were isolated. We anticipate that this simple, rapid method to isolate intact, transient complexes will enable the discoveries of new signaling pathways, biomarkers, and drug targets.

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Section: Resultsmentioning

confidence: 99%

Weak protein–protein interactions revealed by immiscible filtration assisted by surface tension

Berry

Chin

Jackson

et al. 2014

Analytical Biochemistry

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“…mAb 8RB13 was produced in continuous culture, using INTEGRA CELLine CL350 Flasks (distributed by Argo, Elgin, IL) as described (3). Antibody was harvested every 3-4 days by removing the contents of the cell compartment, centrifuging the contents at 1500 rpm, removing the supernatant, and storing it at -20 °C.…”

Section: Methodsmentioning

confidence: 99%

“…These conditions can result in the protein of interest being denatured. The PR-mAb allows for a simple, gentle elution of the protein of interest by using buffers containing low molecular weight polyhydroxylated compounds (polyols) and non-chaotropic salt (1-3). It is currently unknown why some mAbs are polyol-responsive and some are not, but the epitopes for PR-mAbs (designated “softags”) can be used as a way to tag and purify proteins of interest without affecting the protein’s function (4, 5).…”

Section: Introductionmentioning

confidence: 99%

The epitope for the polyol-responsive monoclonal antibody 8RB13 is in the flap-domain of the beta-subunit of bacterial RNA polymerase and can be used as an epitope tag for immunoaffinity chromatography

Stalder

Nagy

Batalla

et al. 2011

Protein Expression and Purification

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Polyol-responsive monoclonal antibodies (PR-mAbs) are useful for the purification of proteins in an easy, one step immunoaffinity step. These antibodies allow for gentle purification of proteins and protein complexes using a combination of a low molecular weight polyhydroxylated compound (polyol) and a nonchaotrophic salt in the eluting buffer. mAb 8RB13 has been characterized as one of these PR-mAbs and has been used to purify RNA polymerase from 5 species of bacteria. Here the epitope for 8RB13 has been identified as PEEKLLRAIFGEKAS, a sequence that is highly conserved in the β-subunit of bacterial RNA polymerase. This sequence is located in the “beta-flap” domain of RNA polymerase (and essentially comprises the “flap-tip helix”), an important binding site for sigma70. This location explains why only the core RNAP is purified using this mAb. This amino acid sequence has been developed into an epitope tag that can be used to purify a target protein from either bacterial or eukaryotic cells when genetically fused to a protein of interest.

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“…Antibody-producing hybridomas were grown in ascites fluid or Celline flasks (IBS Integra Biosciences, Chur, Switzerland) according to manufacturer instructions [28], and mAbs were purified as follows. To remove albumin from IgG 1 antibodies, samples were precipitated with saturated ammonium sulfate (45% w/v), mixed on ice for twenty minutes, then incubated at 4 °C for eighteen hours.…”

Section: Methodsmentioning

confidence: 99%

Production and characterization of monoclonal antibodies to estrogen-related receptor alpha (ERRα) and use in immunoaffinity chromatography

Esch

Thompson

Lamberski

et al. 2012

Protein Expression and Purification

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Estrogen-related receptor alpha (ERRα) is an orphan nuclear receptor whose elevated expression is thought to contribute to breast, colon, and ovarian cancers. In order to investigate the role of ERRα in human disease, there is a need for immunological reagents suitable for detection and purification of ERRα. We expressed recombinant human ERRα in Escherichia coli, purified the protein, and used it to generate monoclonal antibodies (mAbs) to ERRα. Nine high-affinity mAbs were chosen for their abilities to detect overexpressed ERRα in enzyme-linked immunosorbent assays (ELISAs) and Western blots, after which isotyping and preliminary epitope mapping was performed. The mAbs were all IgG subtypes and reacted with several different regions of full-length ERRα. A majority of the mAbs were found to be useful for immunoprecipitation of ERRα, and several could detect DNA-bound ERRα in electrophoretic mobility supershift assays (EMSAs) and chromatin immunoprecipitation (ChIP). The suitability of mAbs to detect ERRα in immunofluorescence assays was assessed. One mAb in particular, 2ERR10, could specifically detect endogenous ERRα in mammary carcinoma cells. Finally, we performed assays to screen for mAbs that gently release ERRα in the presence of a low-molecular-weight polyhydroxylated compound (polyol) and nonchaotropic salt. Using gentle immunoaffinity chromatography, we were able to isolate ERRα from mammalian cells by eluting with a polyol-salt solution. Our characterization studies show that these monoclonal antibodies perform well in a variety of biochemical assays. We anticipate that these novel reagents will prove useful for the detection and purification of ERRα in research and clinical applications.

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Chapter 28 Identification, Production, and Use of Polyol-Responsive Monoclonal Antibodies for Immunoaffinity Chromatography

Cited by 14 publications

References 27 publications

Weak protein–protein interactions revealed by immiscible filtration assisted by surface tension

Weak protein–protein interactions revealed by immiscible filtration assisted by surface tension

The epitope for the polyol-responsive monoclonal antibody 8RB13 is in the flap-domain of the beta-subunit of bacterial RNA polymerase and can be used as an epitope tag for immunoaffinity chromatography

Production and characterization of monoclonal antibodies to estrogen-related receptor alpha (ERRα) and use in immunoaffinity chromatography

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