The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
Use a concentration of 2 µg/ml. Detects a band of approximately 91 kDa (predicted molecular weight: 91 kDa).
SPON1 is a member of a subgroup of the thrombospondin type 1 (TSR) class molecules, defined by two domains of homology, the FS1/FS2 and TSR domains. The TSRs of SPON1 proteins are typical of class 2 TSRs.
SPON1, which is similar to thrombospondin, is a extracellular matrix attached molecule that promotes neurite outgrowth and inhibits angiogenesis. Analysis of gain and loss of function experiments reveal that SPON1 is required for accurate pathfinding of embryonic axons, and plays a dual role in patterning axonal trajectories. It promotes the outgrowth of commissural and inhibits the outgrowth of motor axons, and has also been implicated in inflammatory processes in the nervous system.
Immunohistochemistry (PFA perfusion fixed frozen sections) - Anti-SPON1 antibody (ab40797)This image is courtesy of an abreview submitted by Sophie Pezet, ESPCI, France
IHC-FoFr image of SPON1 staining on Mouse Spinal Cord using ab40797 (1:1000).The sections used came from animals perfused fixed with Paraformaldehyde 4% with 15% of a solution of saturated picric acid, in phosphate buffer 0.1M. Following postfixation in the same fixative overnight, the spinal cord were cryoprotected in sucrose 30% overnight. Spinal cords were then cut using a cryostat and the immunostainings were performed using the ‘free floating’ technique.
All lanes : Anti-SPON1 antibody (ab40797) at 2 µg/ml
Lane 1 : Brain (Rat) Tissue Lysate Lane 2 : Spinal Cord (Rat) Tissue Lysate Lane 3 : Ovary (Mouse) Tissue Lysate Lane 4 : Mouse (14 day old) Fetal Tissue Lysate Lane 5 : Brain (Rat) Tissue Lysate with Human SPON1 peptide (ab41539) at 1 µg/ml Lane 6 : Spinal Cord (Rat) Tissue Lysate with Human SPON1 peptide (ab41539) at 1 µg/ml Lane 7 : Ovary (Mouse) Tissue Lysate with Human SPON1 peptide (ab41539) at 1 µg/ml Lane 8 : Mouse (14 day old) Fetal Tissue Lysate with Human SPON1 peptide (ab41539) at 1 µg/ml
Lysates/proteins at 15 µg per lane.
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 91 kDa
ab40797 detects a 91 kDa band in the rat and mouse tissue lysates shown above. Addition of the immunizing peptide (derived from the SPON1 rat sequence) completely blocks ab40797 in rat tissue, however we are unsure as to why only partial blocking is observed in mouse tissue lysates. Blast analysis of the peptide sequence predicts 100% cross-reactivity with mouse.
ICC/IF image of ab40797 stained PC12 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 (ab40797, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899 Dylight 488 goat anti-rabbit IgG (H+L) used at a 1/250 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.
Port SA et al. The Oncogenic Fusion Proteins SET-Nup214 and Sequestosome-1 (SQSTM1)-Nup214 Form Dynamic Nuclear Bodies and Differentially Affect Nuclear Protein and Poly(A)+ RNA Export. J Biol Chem291:23068-23083 (2016).
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