Production and characterization of isotype-specific monoclonal antibodies to bovine brain Rab GDI.
Small GTPases of the Rab family play a key role in controlling vesicular transport, and the Rab GDP-dissociation inhibitor (GDI) is a regulatory protein for the Rab proteins. Here we report the production and characterization of isotype-specific monoclonal antibodies (MAbs) to Rab GDI. Rab GDI was purified from bovine brain in several steps of column chromatography and was injected into BALB/c mice intraperitoneally. The resulting MAbs specifically recognized a single protein band of 55 kDa, which comigrates with purified bovine Rab GDI. To localize Rab GDI, we processed cells from different sources for indirect immunofluorescence microscopy. Interestingly, the MAb stained cytosol and vesicular structures in brain cells, whereas it predominantly stained cytosol in nonbrain cells. Next, we investigated the cross-reactivities of brain Rab GDI from some mammals. The immunoreactive bands on Western blots appeared to be the same in molecular mass, 55 kDa, in all mammalian species tested including human. In summary, we produced a panel of MAbs that are GDI-alpha/1 form-specific and we believe that the MAbs will be valuable tools in elucidating the function of Rab GDI isoforms.
Ha
S G
SG
Department of Genetic Engineering, Hallym University, Chunchon, Korea.
Park
J B
JB
Ko
K H
KH
Choi
E Y
EY
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology*; D000918:Antibody Specificity; D001923:Brain Chemistry / Q000276:immunology*; D002417:Cattle / Q000276:immunology*; D002478:Cells, Cultured / Q000737:chemistry / Q000276:immunology; D003429:Cross Reactions / Q000276:immunology; D005260:Female; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000032:analysis / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007132:Immunoglobulin Isotypes / Q000276:immunology*; D007150:Immunohistochemistry; D008297:Male; D008322:Mammals / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D020033:Protein Isoforms / Q000032:analysis / Q000276:immunology; D051381:Rats; D013045:Species Specificity
D000911:Antibodies, Monoclonal; C086796:GDP dissociation inhibitor 1; D020730:Guanine Nucleotide Dissociation Inhibitors; D007132:Immunoglobulin Isotypes; D020033:Protein Isoforms