The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/25000 - 1/50000. Detects a band of approximately 90 kDa (predicted molecular weight: 86 kDa).
1/100 - 1/250. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.
Is unsuitable for Flow Cyt or ICC.
Gelsolin is a calcium regulated, actin modulating protein that binds to the plus (or barbed) ends of actin monomers or filaments, preventing monomer exchange (end blocking or capping). It can promote the assembly of monomers into filaments (nucleation) as well as sever filaments already formed. It plays a role in phosphoinositide mediated disassembly of actin filaments in Sertoli cell adhesion complexes; it may regulate sperm release and turnover of the blood testis barrier [RGD], and severs actin filaments in the presence of submicromolar calcium, thereby solating cytoplasmic actin gels. A calcium independent mechanism reverses the process. Northern blot analysis suggested that a single gene encodes both cell and plasma gelsolins. This protein may be unique in that it is made for both secretion and intracytoplasmic location.
Lane 1 : THP-1 (human acute monocytic leukemia) whole cell lysate Lane 2 : THP-1 cultured medium Lane 3 : MCF-7 (human breast adenocarcinoma) whole cell lysate Lane 4 : Human Plasma Lane 5 : THP-1 (human acute monocytic leukemia) whole cell lysate Lane 6 : THP-1 cultured medium Lane 7 : MCF-7 (human breast adenocarcinoma) whole cell lysate Lane 8 : Human Plasma Lane 9 : THP-1 (human acute monocytic leukemia) whole cell lysate Lane 10 : THP-1 cultured medium Lane 11 : MCF-7 (human breast adenocarcinoma) whole cell lysate Lane 12 : Human Plasma
Lysates/proteins at 10 µg per lane.
Secondary Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
The secretory isoform of gelsolin is at 86kDa. The cytoplasmic isoforms of gelsolin are at 81kDa. Lane 5-8: ab109014 detected both secretory and cytoplasmic isoforms. Lane 9-12: ab134183 detected both secretory and cytoplasmic isoforms.
Western blot - Anti-Gelsolin plasma antibody [EPR1941Y] (ab75832)
Anti-Gelsolin plasma antibody [EPR1941Y] (ab75832) at 1/50000 dilution + human plasma at 10 µg
Secondary HRP labelled goat anti-rabbit at 1/2000 dilution
ab75832 Immunoprecipitating Gelsolin plasma in mouse embryo whole cell lysate. 2µg of capture antibody (ab75832) per 0.35mg of lysate was used. 10µg of plasma was incubated with primary antibody (Panel A: ab75832, Panel B: ab134183) at a dilution of 1/1000 and VeriBlot for IP secondary antibody (HRP) ab131366 was used as the secondary antibody at a dilution of 1/10000.
Lane 1: Human plasma Lane 2: Human plasma Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab75832 in Human plasma.
Exposure time: Panel A - 8 seconds, Panel B - 3 minutes.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
The additional band at 50 kDa is a rabbit IgG heavy chain.
Hashimoto K et al. The potential of neurotensin secreted from neuroendocrine tumor cells to promote gelsolin-mediated invasiveness of prostate adenocarcinoma cells. Lab Invest95:283-95 (2015).
Read more (PubMed: 25581609) »
Polioudakis D et al. MiR-191 Regulates Primary Human Fibroblast Proliferation and Directly Targets Multiple Oncogenes. PLoS One10:e0126535 (2015).
Read more (PubMed: 25992613) »