The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Detects a band of approximately 13 kDa (predicted molecular weight: 13 kDa).
Use a concentration of 5 µg/ml.
Use a concentration of 0.1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Calcium-binding protein. Has antimicrobial activity towards bacteria and fungi. Important for resistance to invasion by pathogenic bacteria. Up-regulates transcription of genes that are under the control of NF-kappa-B. Plays a role in the development of endotoxic shock in response to bacterial lipopolysaccharide (LPS) (By similarity). Promotes tubulin polymerization when unphosphorylated. Promotes phagocyte migration and infiltration of granulocytes at sites of wounding. Plays a role as a pro-inflammatory mediator in acute and chronic inflammation and up-regulates the release of IL8 and cell-surface expression of ICAM1. Extracellular calprotectin binds to target cells and promotes apoptosis. Antimicrobial and proapoptotic activity is inhibited by zinc ions.
Expressed by macrophages in acutely inflammed tissues and in chronic inflammation. Detected in peripheral blood leukocytes, in neutrophils and granulocytes. Detected at sites of vascular inflammation (at protein level). Also expressed in epithelial cells constitutively or induced during dermatoses.
Belongs to the S-100 family. Contains 2 EF-hand domains.
Phosphorylated. Phosphorylation inhibits activation of tubulin polymerization.
Secreted. Cytoplasm. Cytoplasm > cytoskeleton. Cell membrane. Associates with tubulin filaments in activated monocytes. Targeted to the cell surface upon calcium influx. Released from blood leukocytes upon exposure to CSF2/GM-CSF, bacterial lipopolysaccharide (LPS) and during inflammatory processes. Serum levels are high in patients suffering from chronic inflammation.
IHC image of S100A9 staining in Human Tonsil formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab63818, 0.1 µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
Western blot - Anti-S100A9 antibody (ab63818)
All lanes : Anti-S100A9 antibody (ab63818) at 1 µg/ml
Lane 1 : Human lymph node tissue lysate - total protein (ab29871) Lane 2 : Human thymus tissue lysate - total protein (ab30146) Lane 3 : Human spleen tissue lysate - total protein (ab29699) Lane 4 : Lung (Human) Tissue Lysate
Lysates/proteins at 10 µg per lane.
Secondary Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution Developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 13 kDa Observed band size : 13 kDa
ICC/IF image of ab63818 stained MCF7 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 (ab63818, 5µg/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. This antibody also gave a positive result in 100% methanol fixed (5 min) HeLa, HepG2 and Hek293 cells at 5µg/ml, and in 4% PFA fixed (10 min) HeLa, HepG2, Hek293 and MCF7 cells at 5µg/ml.
Dey J et al. A Platform for Rapid, Quantitative Assessment of Multiple Drug Combinations Simultaneously in Solid Tumors In Vivo. PLoS One11:e0158617 (2016).
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