IIB   20738
INSTITUTO DE INVESTIGACIONES BIOLOGICAS
Unidad Ejecutora - UE
artículos
Título:
An alternative easy method for antibody purification and protein-protein interactions analyzes using GST fusion proteins immobilized onto glutathione agarose
Autor/es:
L. ZALAZAR; C.A.I. ALONSO; R.E. DE CASTRO; A. CESARI
Revista:
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
Editorial:
SPRINGER HEIDELBERG
Referencias:
Lugar: HEIDELBERG; Año: 2014 vol. 406 p. 911 - 914
ISSN:
1618-2642
Resumen:
Abstract 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 compromises the interaction between the protein
and its target. In these cases, glutathione-S-transferase
(GST) is a valuable tag providing a long arm that makes
the bait protein accessible to the mobile flow phase of the
chromatography. In the present report, we used a GST
fusion version of the 8-kDa protein serine protease inhibitor
Kazal-type 3 (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
(dithiobis(succinimidyl propionate)) 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 reuse of the column for alternative
uses, such as screening for other protein?protein interactions
using SPINK3 as the bait.