Abstract

 Optimization of Protein Coffee Leaf Quantification
by Fluorescent Immunodetection Method
E.A. SOARES, S.H.C. ARAÚJO, C.Q.G. SOARES, A.M. ALMEIDA, M.E. LOUREIRO
Departamento de Biologia Vegetal, Universidade Federal de Viçosa, Viçosa – MG, Brazil.
E-mail: [email protected]
SUMMARY
Protein immunodetection (Western blotting) is an important tool to dissect the function of a
gene and physiological processes. In this work, we used a labeled secondary antibody with
fluorophore (IgG – Cy5TM; goat – α – rabbit). The fluorescence was scanned by scanner
(FLA-3000), and the image was analyzed by image quantitative analyzer software (Multi
Gauge). Total coffee leaf protein from two Coffea canephora clones with contrasting drought
tolerance, was extracted through phenol buffer method and separated by SDS-PAGE. The
proteins were transferred to PVDF membranes using a semi-dry apparatus. Specific primary
antibody against photosystem II D1 protein and Ribulose-1,5-biphosphate carboxylaseoxidase large subunit (RbcL) were used. These proteins play central role in key steps of
photochemical and biochemical pathways of photosynthesis. Both proteins were detected as
sharp bands emitting strong fluorescent signal in the imunoblottings, highest signal was
obtained using chemioluminescent detection method (CDP-Star, GE), or colorimetric
detection method (NBT + BCIP).