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Synthetic peptide conjugated to KLH derived from within residues 750 to the C-terminus of Human Smoothened.
(Peptide available as ab39344.)
Our Abpromise guarantee covers the use of ab38686 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use a concentration of 1 µg/ml. Predicted molecular weight: 86 kDa.|
|ICC/IF||Use a concentration of 5 µg/ml.|
Immunohistochemistical detection of Smoothened protein with antibody ab38686 on rat brain PFA-perfusion fixed frozen sections. Primary antibody ab38686 was diluted 1/3000 and incubated for 18 hours @ 20°C in PBS + 0.3 % TritonX100. Secondary antibody: goat anti-rabbit Alexa Fluor 488® (1/1000). This antibody was used on (30um) free-floating sections of rat brain. The antibody produced some staining in the whole CNS, in glial cells as well as some diffuse staining. No staining was observed in the dentate gyrus although this was expected as Rajendran et al (Neurosci Lett. 2009 April 10 453(3): 190–194) showed the presence of Smoothened mRNA in this structure. However, literaure on the protein expression is very limited; it is possible that the protein might be expressed in gial cells too. The pictures submitted show the staining at the level of the striatum (top panel). ab38686 stains some neuron-like cells and some glial cel
NIH-3T3 cells were incubated at 37°C for 7h with vehicle control (0 µM) and 5 µM of jervine (ab120815) in DMSO. Decreased expression of smoothened (ab38686) in NIH-3T3 cells correlates with an increase in jervine concentration, as described in literature.
Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 20µg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 3% milk before being incubated with ab38686 at 1 µg /ml and ab8227 at 1 µg /ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.