Human PRELP Antibody

  
  • Species Reactivity
    Human
  • Specificity
    Detects human PRELP in direct ELISAs and Western blots.
  • Source
    Polyclonal Sheep IgG
  • Purification
    Antigen Affinity-purified
  • Immunogen
    Chinese hamster ovary cell line CHO-derived recombinant human PRELP
    Gln21-Ile382
    Accession # P51888
  • Formulation
    Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied as a 0.2 µm filtered solution in PBS.
  • Endotoxin Level
    <0.10 EU per 1 μg of the antibody by the LAL method.
  • Label
    Unconjugated
Applications
  •  
    Recommended
    Concentration
    Sample
  • Western Blot
    1 µg/mL
    See below
  • Immunocytochemistry
    5-15 µg/mL
    See below
  • Neutralization
    Measured by its ability to neutralize PRELP inhibition of TRANCE/TNFSF11/RANK L-induced osteoclast-like cell formation in the RAW 264.7 mouse monocyte/macrophage cell line. Rucci, N. et al. (2009) J. Cell Biol. 187:669. The Neutralization Dose (ND50) is typically 0.2-1.2 µg/mL in the presence of 5 µg/mL Recombinant Human PRELP, 5 ng/mL Recombinant Mouse TRANCE/TNFSF11/RANK L, and 20 ng/mL Recombinant Mouse M‑CSF.
Please Note: Optimal dilutions should be determined by each laboratory for each application. General Protocols are available in the Technical Information section on our website.
Data Examples
PRELP Inhibition of TRANCE-dependent Osteoclast-Like Cell Formation and Neutralization by Human PRELP Antibody.

Recombinant Human PRELP (Catalog # 6447-PR) inhibits Recombinant Mouse TRANCE induced osteoclast-like cell formation in the RAW 264.7 mouse monocyte/macrophage cell line in a dose-dependent manner (orange line), as measured by the TRAP (tartrate‑resistant acid phosphatase) solution assay. Inhibition of Recombinant Mouse TRANCE (5 ng/mL) activity elicited by Recombinant Human PRELP (5 µg/mL) is neutralized (green line) by increasing concentrations of Sheep Anti-Human PRELP Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6447). The ND50 is typically 0.2-1.2 µg/mL in the presence of Recombinant Mouse M‑CSF (20 ng/mL).

Detection of Human PRELP by Western Blot. Western blot shows lysates of human liver tissue and human pancreas tissue. PVDF membrane was probed with 1 µg/mL of Sheep Anti-Human PRELP Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6447) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specific band was detected for PRELP at approximately 65 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.
Immunocytochemistry
PRELP in Human Mesenchymal Stem Cells. PRELP was detected in immersion fixed human mesenchymal stem cells differentiated into chondrocytes using Sheep Anti-Human PRELP Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6447) at 10 µg/mL overnight at 4 °C. 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.
Preparation and Storage
  • Reconstitution
    Sterile PBS to a final concentration of 0.2 mg/mL.
  • Shipping
    The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below. *Small pack size (SP) is shipped with polar packs. Upon receipt, store it immediately at -20 to -70 °C
  • Stability & 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.
Background: PRELP

PRELP (Proline aRginine-rich End Leucine-rich repeat Protein; also Prolargin) is a 55‑62 kDa secreted glycoprotein that belongs to the small leucine-rich proteoglycan (SLRP) superfamily of extracellular matrix (ECM) molecules (1‑4). Within this family, it is considered a class II member, implying that it is unlikely to form dimeric structures (3). PRELP is synthesized as a 382 amino acid (aa) precursor that contains a 20 aa signal sequence plus a 362 aa mature region (1, 5). Like other SLRPs, PRELP contains an N-terminal extension (aa 72‑107) coupled to multiple Leu-rich repeats (LRRs) (aa 95‑382) (6). Unlike other SLRPs, PRELP does not contain any proteoglycan chains, and its N‑terminal extension is highly basic in charge. The N-terminus reportedly binds to negatively-charged heparin/heparin-sulfate, chondroitin sulfate, and Gram- bacterial cell walls, while the LRR region participates in protein-protein interactions (7‑9). Although PRELP is known to be synthesized by only a few cell types, including osteoblasts, skeletal muscle and chondrocytes, its expression is likely to be more widespread, given its presence in the basement membrane (BM) of Bowman’s capsule, epididymal epithelium and the stratified squamous epithelium of the skin (1, 10, 11). The dual binding profile of PRELP is key to its function. In cartilage, PRELP likely links chondrocyte cell membrane heparin sulfate (HS) chains to endogenous type II collagen. Within the context of the BM, PRELP likely plays an anchoring role. The BM is composed of type IV collagen and laminin, linked together by nidogen. BM Perlecan reinforces this linkage by binding to all three components. PRELP, on the edge of the BM, can bind to free perlecan HS chains (via its N-terminus), and to underlying type I collagen (via its LRRs), thus forming an anchor for the BM (11). Notably, the N-terminus appears to do more than simply provide part of a linkage mechanism. In bone, osteoblast secreted PRELP is hypothesized to undergo proteolysis by enzymes such as LysC and glutamyl endopeptidase. This will generate 40‑75 aa N‑terminal fragments that can bind to chondroitin sulfate adducts that exist on the surface of prefusion osteoclast precursors. Following binding, PRELP is internalized, complexed to annexin-II, and translocated to the nucleus, where it interacts with NF kappa Bp65 to block osteoclast maturation (8). In tissue, PRELP may also undergo proteolytic processing during inflammation to release an N‑terminal fragment containing aa 21‑42 of the precursor (7). This sequence has been shown to possess potent antimicrobial activity by creating pores in bacterial cell walls. Mature human PRELP shares 91% aa identity with mouse PRELP (10).

  • References:
    1. Bengtsson, E. et al. (1995) J. Biol. Chem. 270:25639.
    2. Merline, R. et al. (2009) J. Cell Commun. Signal. 3:323.
    3. McEwan, P.A. et al. (2006) J. Struct. Biol. 155:294.
    4. Neame, P.J. et al. (1999) Cell. Mol. Life Sci. 55:1327.
    5. Grover, J. et al. (1996) Genomics 38:109.
    6. SwissProt # P51888.
    7. Bengtsson, E. et al. (2000) J. Biol. Chem. 275:40695.
    8. Rucci, N. et. al. (2009) J. Cell Biol. 187:669.
    9. Malmsten, M. et al. (2006) Matrix Biol. 25:294.
    10. Grover, J. & P.J. Roughley (2001) Matrix Biol. 20:555.
    11. Bengtsson, E. et al. (2002) J. Biol. Chem. 277:15061.
  • Long Name:
    Proline-arginine-Rich End Leucine-rich repeat Protein
  • Entrez Gene IDs:
    5549 (Human)
  • Alternate Names:
    55 kDa leucine-rich repeat protein of articular cartilage; MST161; MSTP161; PRELP; prolargin proteoglycan; Prolargin; proline arginine-rich end leucine-rich repeat protein; proline/arginine-rich end leucine-rich repeat protein; Proline-arginine-rich end leucine-rich repeat protein; SLRR2A; SLRR2AMGC45323
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