POLYCLONAL POLYCLONAL ANTIBODIES TO THE RECOMBINANT LST8/GΒL HOMOLOGUE OF TRITICUM AESTIVUM, A COMPONENT OF THE TORC1 SIGNALING
DOI:
https://doi.org/10.26577/eb.2021.v88.i3.10Keywords:
Triticum aestivum, LST8, protein expression, polyclonal antibodies, enzyme immunoassay.Abstract
LST8 is a key component of the TOR signaling system, a central regulator of cell proliferation and growth in eukaryotic cells. In this study, polyclonal antibodies against LST8 of Triticum aestivum (TaLST8) were produced and characterized. cDNA of the wheat TaLST8 was isolated by reverse transcription-PCR. The amino acid sequence alignment of TaLST8 showed high homology with yeast LST8 (55% identity), human LST8 (51% identity), and Arabidopsis AtLST8 (85% identity). To obtain antibodies against TaLST8, a highly conserved part of the TaLST8 gene encoding a 150 amino acid protein (talst8/150) was cloned into pET-28c with a histidine tag (6xHis) and expressed in Escherichia coli. Rabbits were immunized with purified TaLST8/150 recombinant protein. The obtained antisera were purified to increase the specificity of recognition. The sensitivity and specificity of the produced antibody was analyzed by enzyme-linked immunosorbent assay, Western blotting and immunodot assay. Enzyme-linked immunosorbent assay showed that immunization with purified 6xHis-TaLST8/150 produced the high titer (1:64000) polyclonal antibodies with high specificity. Antibodies against TaLST8 allowed the sensitive detection of native and denatured protein in Western blot and immunodot assays.
The purified polyclonal antibody raised against the recombinant TaLST8/150 protein is sufficiently specific and sensitive and could be a useful tool for future insights of the functioning of the TOR signaling system in wheat plants.
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