Primary antibody notesThe dynamins are a family of 100 kDa GTPases transcribed from at least three separate genes. At least four mRNA splice variants for each dynamin have been described. Dynamins contain several conserved regions including the conserved, amino-terminal GTPase domain, a centrally located membrane-binding plekstrin homology domain (PHD), and a coiled-coil region located in front of a proline-rich domain (PRD). The PRD is thought to mediate interactions between dynamin and numerous other cellular proteins. Dynamin 1 is expressed exclusively in neurons, Dynamin 2 is ubiquitously expressed, and Dynamin 3 is thought to be restricted to expression in the brain, testis, heart, and lung. The dynamins participate in the cellular process of clathrin-mediated and fluid-phase endocytosis.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration. Can be blocked with Dynamin 3 peptide (ab4986). By Western blot, this antibody detects an ~100 kDa protein representing Dynamin 3 from HeLa cell lysate.
Use a concentration of 4 µg/ml.
Use a concentration of 1 µg/ml.
FunctionMicrotubule-associated force-producing protein involved in producing microtubule bundles and able to bind and hydrolyze GTP. Most probably involved in vesicular trafficking processes, in particular endocytosis.
Sequence similaritiesBelongs to the TRAFAC class dynamin-like GTPase superfamily. Dynamin/Fzo/YdjA family. Contains 1 dynamin-type G (guanine nucleotide-binding) domain. Contains 1 GED domain. Contains 1 PH domain.
ab3458 (4µg/ml) staining Dynamin 3 in human brain cerebellum using an automated system (DAKO Autostainer Plus). Using this protocol there is strong staining of nuclear/cytoplasmic compartments within the white matter region Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH 6.1 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
ICC/IF image of ab3458 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab3458, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
Hyndman KA et al. Dynamin activates NO production in rat renal inner medullary collecting ducts via protein-protein interaction with NOS1. Am J Physiol Renal Physiol301:F118-24 (2011).
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