Immunodetection of mitochondrial glycerophosphate dehydrogenase (mGDH) by a polyclonal antibody raised against a recombinant mGDH fragment product.
The mitochondrial enzyme glycerophosphate dehydrogenase (mGDH) plays an essential role in the B-cell glucose-sensing device and its activity in islet homogenates is impaired in several animal models of type 2 diabetes. We have now developed a polyclonal antibody, raised against a recombinant mGDH fragment product, that could be used for the immunodetection of mGDH. Total RNA was isolated from rat pancreatic islets and used in the synthesis of cDNA. Specific primers were designed that corresponded to the FAD binding domain of mGDH. The PCR product was purified and cloned into an appropriate expression vector used for transformation of Escherichia coli cells. The fusion protein was extracted from the transformed cells, further purified, and used for immunization of rabbits. The antibody recognized a single band of 72 kDa in rat islets and testis. The recombinant mGDH product was also recognized as a single band with the expected 65-kDa reference. An ELISA procedure was designed for detection of antibodies against the recombinant mGDH fragment product. The availability of the mGDH antibody opens the way to a number of further applications such as immunocytochemis- try and mGDH quantification in biological material.
Novials
A
A
Endocrinology Unit and Service of Immunology, Barcelona University, Barcelona, Spain.
Fabregat
M E
ME
Benito
C
C
Fernandez-Alvarez
J
J
Gallart
T
T
Malaisse
W J
WJ
Gomis
R
R
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D003922:Diabetes Mellitus, Type 1 / Q000378:metabolism; D005993:Glycerolphosphate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D010446:Peptide Fragments / Q000276:immunology*; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000276:immunology*
D000906:Antibodies; D010446:Peptide Fragments; D011994:Recombinant Proteins; D005993:Glycerolphosphate Dehydrogenase