S100A9 Antibody [Unconjugated]

Western blot shows lysates of human peripheral blood mononuclear cells (PBMC), human spleen tissue, human tonsil tissue, and human cartilage tissue. PVDF membrane was probed with 0.2 µg/mL of Sheep Anti-Human S100A9 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF5578) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specific band was detected for S100A9 at approximately 14 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

S100A9 was detected in immersion fixed MDA-MB-468 human breast cancer cell line using Sheep Anti-Human S100A9 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF5578) at 1.7 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Sheep IgG Secondary Antibody (red; Catalog # NL010) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

S100A9 was detected in immersion fixed paraffin-embedded sections of human cartilage using Sheep Anti-Human S100A9 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF5578) at 1 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Sheep HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS019) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm of chondrocytes. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Western blot shows lysates of MDA-MB-468 human breast cancer parental cell line and S100A9 knock out MDA-MB-468 cell line (KO). PVDF membrane was probed with 0.2 µg/mL of Sheep Anti-Human S100A9 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF5578) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specific band was detected for S100A9 at approximately 14 kDa (as indicated) in the parental MDA-MB-468 cell line, but is not detectable in knockout MDA-MB-468 cell line. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Human peripheral blood monocytes (PBMC) were stained with Mouse Anti-Human CD14 PE-conjugated Monoclonal Antibody (Catalog # FAB3832P) and either (A) Sheep Anti-Human S100A9 Polyclonal Antibody (Catalog # AF5578) or (B) Sheep IgG Isotype Control (Catalog # 5-001-A) followed by anti-Sheep IgG APC-conjugated Secondary Antibody (Catalog # F0127). To facilitate intracellular staining, cells were fixed with Flow Cytometry Fixation Buffer (Catalog # FC004) and permeabilized with Flow Cytometry Permeabilization/Wash Buffer (Catalog # FC005). View our protocol for Staining Membrane-associated Proteins.

S100A9 was detected in immersion fixed MDA-MB-468 human breast cancer cell line but is not detected in S100A9 knockout (KO) MDA-MB-468 cell line using Sheep Anti-Human S100A9 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF5578) at 1.7 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Sheep IgG Secondary Antibody (red; Catalog # NL010) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

• Reactivity: Hu
• Applications: WB, IHC, Flow, ICC/IF, KO
• Clonality: Polyclonal
• Host: Sheep
• Conjugate: Unconjugated
• Concentration: LYOPH

S100A9 Antibody [Unconjugated] Summary

• Immunogen: E. coli-derived recombinant human S100A9
  The2-Pro114
  Accession # P06702
• Specificity: Detects human S100A9 in direct ELISAs and Western blots. In direct ELISAs, less than 5% cross-reactivity with recombinant mouse S100A9 is observed.
• Source: N/A
• Isotype: IgG
• Clonality: Polyclonal
• Host: Sheep
• Gene: S100A9
• Purity Statement: Antigen Affinity-purified

Applications/Dilutions

• Dilutions:
  • Immunocytochemistry 1-25 ug/mL
  • Immunohistochemistry 5-15 ug/mL
  • Intracellular Staining by Flow Cytometry 0.25 ug/10^6 cells
  • Knockout Validated
  • Western Blot 0.2 ug/mL

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.
• Preservative: No Preservative
• Concentration: LYOPH
• Reconstitution Instructions: Reconstitute at 0.2 mg/mL in sterile PBS.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for S100A9 Antibody [Unconjugated]

• CAGBMigration inhibitory factor-related protein 14
• Calgranulin B
• calgranulin-B
• Calprotectin L1H subunit
• CFAGMRP-14,60B8AG
• CGLB
• L1AG
• LIAG
• MAC387
• MIF
• MRP-14
• MRP14Leukocyte L1 complex heavy chain
• NIF
• P14
• protein S100-A9
• S100 calcium binding protein A9 (calgranulin B)
• S100 calcium binding protein A9
• S100 calcium-binding protein A9 (calgranulin B)
• S100 calcium-binding protein A9
• S100A9

Background

Human S100A9 (also MRP-14, Calgranulin-B, and p14) is a 14 kDa member of the S100 family of EF-hand calcium-binding proteins.  It is 114 amino acids (aa) in length and contains short sequential modules.  There is an N-terminal helical region, followed by a calcium-binding EF-hand domain, two more helical regions, a second EF-hand domain, and three additional helical regions.  S100A9 will noncovalently heterodimerize with S100A8.  In the presence of calcium, this heterodimer will form a heterotetramer. S100A9 is expressed in granulocytes, monocytes, and macrophages during acute and chronic inflammation.  Human S100A9 shares 62% and 57% aa identity with rat and mouse S100A9, respectively.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

FAQs for S100A9 Antibody (AF5578). (Showing 1 - 2 of 2 FAQ).

1. Have any of your S100A9 antibodies been tested for use in neutralization, or blocking assays, on human samples?
   • Unfortunately at this time we have not tested, or received customer feedback on our S100A9 antibodies for use in blocking or neutralizing assays. If you were planning on testing any of our S100A9 antibodies for neutralizing or blocking capabilities, we would recommend our Innovator’s Reward Program. Under the terms of this program we would be happy to provide a credit for a free vial in exchange for new data on this previously untested or reported application. Please submit this data in the form of an online review. Additional <a href="http://www.novusbio.com/support/innovators-reward.html" target="_self">Innovators Reward Program</a> information can be found using this link.
2. We would like to stain paraffin embedded human intestinal tissue for Calprotectin. One question will be which cells express the majority of Calprotectin - invading neutrophils vs epithelium for example. The antibody list you provide is quite extensive. Which antibody would you recommend for our purpose? Which tissue would you recommend as positive control.
   • Nine of our antibodies to Calprotectin have been validated for IHC-P with human tissue, and you can see all of these products. A number of our IHC images were generated using human spleen samples, and as such this may be a good choice of a positive control tissue. We do sell a human spleen slide product, which is suitable for IHC-P. Unfortunately I do not have an in-depth knowledge of Calprotectin, however the image shown for our antibody with catalogue number NBP1-02826 demonstrates clear staining of neutrophils. The following paper also suggests that the protein is abundant in neutrophils: PMID 11435495.

Bioinformatics

• Gene Symbol: S100A9
• Uniprot:
  • P06702()