Fractionation and Characterization of Polyclonal Antibodies Using Three Progressively More Chaotropic Solvents

Narhi, Linda O.; Caughey, D.J.; Horan, Thomas P.; Kita, Yoshiko; Chang, David C.; Arakawa, Tsutomu

doi:10.1006/abio.1997.2376

Cited by 26 publications

(8 citation statements)

References 10 publications

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“…Polyclonal antisera against heterologously expressed atToc33 and atToc34 protein [13] as well as enhanced green fluorescent protein (EGFP) were raised in rabbits by standard procedures [20]. Affinity purified antibodies were obtained from the sera with NHS‐activated HiTrap columns (Amersham Bioscience) to which the respective antigenes had been coupled [21]. Specificity of the purified antibodies was tested by Western analyses.…”

Section: Methodsmentioning

confidence: 99%

At least two Toc34 protein import receptors with different specificities are also present in spinach chloroplasts

et al. 2005

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The receptor components of the chloroplast protein import machinery, Toc34 and Toc159, are both encoded by small gene families in Arabidopsis thaliana. Recent results suggest that each member of these families preferentially interacts with different groups of precursor proteins. Here we address the question, whether multiple homologous Toc receptors are unique to Arabidopsis or whether they are a general phenomenon in plants. Indeed, in spinach we could identify at least two Toc34 proteins with different substrate specificities as demonstrated by competition and antibody inhibition experiments. In addition, an analysis of the available genomic data revealed the presence of at least two Toc34 homologs in six other plant species.

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Section: Methodsmentioning

confidence: 99%

At least two Toc34 protein import receptors with different specificities are also present in spinach chloroplasts

et al. 2005

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“…Even if the extracted antibodies refold in their native conformation because of immediate neutralisation and/or dialysis following elution (Narhi et al, 1997a(Narhi et al, , 1997b we did not investigate the effect of the elution buffers on their conformation and so cannot exclude that an irreversible denaturation occurred. Nevertheless, our 280 nm absorption measurements of the column eluates indicated that those fractions which lacked anti-LPS antibodies by ELISA also lacked measurable protein content and thus were unlikely to contain significant amounts of denatured antibody.…”

Section: Discussionmentioning

confidence: 98%

“…Glycine at acidic pH is commonly used as an elution buffer (Narhi et al, 1997a(Narhi et al, , 1997b. We therefore tested 0.1 M glycine, 0.1 M NaCl at pH 3, pH 2.8, pH 2.6 and pH 2.4 (Fig.…”

Section: Elution Under Acidic Conditions Is Optimalmentioning

confidence: 99%

Purification of antibodies to O antigen of Salmonella Typhimurium from human serum by affinity chromatography

O’Shaughnessy

Micoli

Gavini

et al. 2013

Journal of Immunological Methods

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Nontyphoidal Salmonellae (NTS) are a common cause of bacteraemia in children and HIV-infected adults in Sub-Saharan Africa. We have previously shown that antibodies play a key role in both bactericidal and cellular mechanisms of immunity to NTS, but found that high concentrations of antibody to Salmonella Typhimurium O antigen (OAg) in the serum of some HIV-infected African adults is associated with impaired killing of NTS. To further investigate the function of antibodies to the OAg of NTS, we developed a method to purify these antibodies from human serum by affinity chromatography. Purified Salmonella Typhimurium OAg was activated with adipic acid dihydrazide (ADH) via two different chemistries before linking to N-hydroxysuccinamide-Sepharose resin: one ADH molecule was introduced per OAg chain on its terminal 3-deoxy-D-manno-octulosonic acid sugar (OAg-ADH), or multiple ADH molecules were attached along the OAg chain after oxidation with sodium periodate (OAgoxADH). Both resulting columns worked well when tested with commercial polyclonal anti-O:4,5 antibodies from rabbit serum. Over 90% of the applied antibodies bound to the resin and 89% of these antibodies were then eluted as detected by ELISA. OAg-ADH was preferred as the method for OAg derivatisation as it does not modify the saccharide chain and can be applied to OAg from different bacteria. Both columns were able to bind OAg-specific antibodies in human serum, but antibody recovery was initially low. Different elution buffers were tested and different amounts of OAg-ADH were linked to the resin to improve the yield. Optimal recovery (51%) was obtained by loading 1mg of activated OAg per ml of resin and eluting with 0.1M glycine, 0.1M NaCl pH2.4. The column matrix could be regenerated following elution with no detectable loss in performance for over ten uses. This method offers the potential to purify antibodies to Salmonella OAg from polyclonal serum following vaccination or natural exposure to Salmonella and so investigate the functionality and diversity of the antibody response to OAg.

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“…(2) a glycine/SDS solution at a low pH (Bolognesi et al, 2017;Gut et al, 2018;Nakane, 1968;Narhi et al, 1997a;Pirici et al, 2009;Sorrelle et al, 2019) 2014) which reduces the disulfide bonds present in antibodies, thus breaking down their tertiary structure (Capel et al, 1980;Crivianu-Gaita et al, 2015); (5) a low-pH oxidizing solution of KMnO 4 and H 2 SO 4 (Glass et al, 2009;Tramu et al, 1978); (6) chaotropic salts (Bolognesi et al, 2017;Gut et al, 2018;Narhi et al, 1997a;Narhi et al, 1997b); (7) combining antibodies with drastically different abundances (Wang et al, 1999).…”

Section: Literature Review and Selection Of Candidate Stripping Technmentioning

confidence: 99%

Multiplex immunofluorescence methods in neurodegenerative diseases

Ehrenberg

Morales

Tejedor

et al. 2019

Preprint

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BACKGROUND: Neurodegenerative diseases feature stereotypical deposits of protein aggregates that selectively accumulate in vulnerable cells. The ability to simultaneously localize multiple targets in situ would facilitate discovery and validation of molecular pathways underlying neurodegenerative diseases.Immunostaining methods offer in situ detection of targets. Most proposed protocols for multiplexing immunostaining require sophisticated equipment not available for standard labs. Effective stripping of antibodies, allowing successive rounds of staining while maintaining tissue and antigen integrity, is the main roadblock for enabling multiplex immunostaining in standard labs. Antibody stripping techniques have not been fully validated for neurodegenerative disease research. Moreover, despite the increased complexity of multiplex immunostaining protocols, quality control steps have not been regularly implemented.NEW METHOD: Aiming to create protocols for multiplex localization of neurodegenerative-related processes, without the need for specialized equipment, we evaluated several antibody stripping techniques. We also recommend quality control steps to validate stripping efficacy and ameliorate concerns of cross-reactivity and false positives. RESULTS:The proposed protocol using β -mercaptoethanol enables reliable antibody stripping across multiple rounds of staining and minimizes the odds of cross-reactivity while preserving tissue and antigen integrity.COMPARISON WITH EXISTING METHODS: Our proposed method accounts for the intricacies of suboptimal human post-mortem tissue and the need to strip markers bound to highly aggregated proteins.Additionally, it incorporates quality control steps to validate antibody stripping.CONCLUSIONS: Multiplex immunofluorescence methods for studying neurodegenerative diseases in human postmortem tissue are feasible even in standard laboratories. Nevertheless, evaluation of stripping parameters in optimization and validation phases of experiments is prudent.

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Fractionation and Characterization of Polyclonal Antibodies Using Three Progressively More Chaotropic Solvents

Cited by 26 publications

References 10 publications

At least two Toc34 protein import receptors with different specificities are also present in spinach chloroplasts

At least two Toc34 protein import receptors with different specificities are also present in spinach chloroplasts

Purification of antibodies to O antigen of Salmonella Typhimurium from human serum by affinity chromatography

Multiplex immunofluorescence methods in neurodegenerative diseases

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