The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application notesICC: Use at an assay dependent dilution.
WB: >1/500. Detects a band of approximately 63 kDa
Not tested in other applications.
Optimal dilutions/concentrations should be determined by the end user.
RelevanceFruit fly (Drosophila melanogaster) ovaries contains two set of germline stem cells surrounded by a group of highly differentiated somatic cells that express genes for two phenotypes (hedgehog and wingless). The TGF beta super family member, decpentaplegic (dpp) or its homologue BMP2/4 is specifically required for maintenance and promotes its cell division in the female germline. The Signaling by TGF beta related factors requires ligand induced association between type I and type II transmembrane receptors that have endogenous serine/threonine kinases activity. In Drosophila, the type I receptor is encoded by the thick veins (tkv) gene and the type II receptor is encoded by the punt (put) gene. These receptors mediate signaling by decapentaplegic (dpp), a member of the bone morphogenetic protein (BMP) subgroup of TGF beta type factors. Over expression or mutation in dpp suppress germline stem cell differentiation. The Drosophila punt gene encodes a type II serine/threonine kinase TGF beta/Dpp (Decapentaplegic) receptor. Dpp actions are mediated by its receptor Punt and Saxophone. There are 5 down stream component in the dpp signaling cascade required to block the development of various organelles including salivary glands. These are Mothers against dpp (Mad), Medea (Med) and Schnurri (Shn). Punt signaling is also responsible for calcium gradient formation during D. melanogaster development. Punt gene encodes for a homolog of vertebrate type II receptor and Punt, like thick veins (Tkv) is essential for in vivo dpp dependent patterning process. No Dpp dependent signal processing is apparent in the absence of Punt ot Tkv suggesting that both receptors acts in concert to transduce Dpp signaling.
Western blot - Anti-Punt antibody (ab14680)Image courtesy of Dr. Richelle Sopko, Harvard Medical School, U.S.
All lanes : Anti-Punt antibody (ab14680) at 1/1000 dilution
Lane 1 : 0-4 hour old EGFP shRNAi embryos (Control) Lane 2 : 0-4 hour old put shRNAi embryos
Lysates/proteins at 30 µg per lane.
Secondary Amersham ECL anti-Rabbit IgG, HRP conjugated at 1/1000 dilution Developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 62 kDa Observed band size : 65 kDa (why is the actual band size different from the predicted?) Additional bands at : 180 kDa (possible non-specific binding),38 kDa (possible non-specific binding),40 kDa (possible non-specific binding),55 kDa (possible non-specific binding),90 kDa (possible non-specific binding).
Exposure time : 4 minutes
Image courtesy of Dr. Richelle Sopko, Harvard Medical School, U.S.