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Title

Affinity Chromatographic Purification of Antibodies to a Biotinylated Fusion Protein Expressed inEscherichia coli

AuthorsAlché Ramírez, Juan de Dios; Dickinson, Hugh
KeywordsChromatography
Affinity purification
HOP1
Fusion protein
Yeast
Antibodies
Issue DateFeb-1998
PublisherElsevier
CitationProtein Expression and Purification 12(1): 138-143 (1998)
AbstractHOP1,a protein component of the synaptonemal complex inSaccharomyces cerevisiaewhich is believed to play an important role in meiotic synapsis, was expressed inEscherichia colias a fusion protein incorporating a “tag” polypeptide which is biotinylated naturally in the bacteria. TheHOP1fusion protein was produced in an insoluble form within the bacteria; once solubilized using a denaturing agent, the protein was purified by avidin monomer affinity chromatography. The recombinant protein was used to immunize rabbits and produce polyclonal antibodies. Procedures for affinity purification of antibodies using the recombinant protein attached to the avidin column and a magnetic method for concentration of antibodies are described. Antibody elution conditions in these procedures do not affect the affinity of the column for the recombinant protein, which can be recovered afterward. Affinity-purified antibodies show high binding capacity toHOP1recombinant protein in immunoblotting experiments, but reduced background compared with crude antiserum or purified IgG fraction. The affinity-purified antibodies recognize a major band around 70 kDa in Western blots of yeast protein extracts following meiotic induction.
Description6 páginas, 4 figuras, 1 tabla.
Publisher version (URL)http://dx.doi.org/10.1006/prep.1997.0824
URIhttp://hdl.handle.net/10261/32701
DOI10.1006/prep.1997.0824
ISSN1046-5928
Appears in Collections:(EEZ) Artículos
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