An alternative easy method for antibody purification and analysis of protein-protein interaction-using GST fusion proteins immobilized onto glutathione-agarose

Authors
Zalazar, Lucia; Alonso, Carlos Agustín Isidro; de Castro, Rosana Esther; Cesari, Andreina
Publication Year
2014
Language
English
Format
article
Status
Published version
Description
Immobilization of small proteins designed to perform protein-protein assays can be a difficult task. Often, the modification of reactive residues necessary for the interaction between the immobilized protein and the matrix compromise the interaction between the protein and its target. In these cases, GST is a valuable tag providing a long arm that makes the bait protein to be accessible to the mobile flow phase of the chromatography. In the present report, we used a GST-fusion version of the 8 kDa protein SPINK3 as the bait to purify anti-SPINK3 antibodies from a rabbit crude serum. The protocol for immobilization of GST-SPINK3 to glutathione agarose beads was modified from previously reported protocols by using an alternative bifunctional crosslinker (DSP) in a very simple procedure and by using simple buffers under physiological conditions. We concluded that the immobilized protein remained bound to the column after elution with low pH, allowing the re-use of the column for alternative uses, such as screening for other protein-protein interactions using SPINK3 as bait.
Fil: Zalazar, Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina
Fil: Alonso, Carlos Agustín Isidro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina
Fil: de Castro, Rosana Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina
Fil: Cesari, Andreina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina
Subject
antibody purification
protein-protein interactions
glutathione-agarose column
DSP cross-linker
SPINK3
Biología Reproductiva
Ciencias Biológicas
CIENCIAS NATURALES Y EXACTAS
Access level
Restricted access
License
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repository
CONICET Digital (CONICET)
Institution
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identifier
oai:ri.conicet.gov.ar:11336/13285