Detection of Human Legumain/ Asparaginyl Endopeptidase by Western Blot. Western blot shows lysates of human kidney tissue, human heart tissue, and human placenta tissue. PVDF membrane was probed with 1 µg/mL of Goat Anti-Human Legumain/Asparaginyl Endopeptidase Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2199) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). Specific bands were detected for Pro-Legumain/ Asparaginyl Endopeptidase at approximately 56 kDa and mature Legumain/Asparaginyl Endopeptidase 37 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.
Preparation and Storage
Reconstitute at 0.2 mg/mL in sterile PBS.
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: Legumain/Asparaginyl Endopeptidase
Legumain is a lysosomal cysteine protease whose activity is found in several tissues tested (1, 2). Legumain plays a pivotal role in the endosomal/lysosomal degradation system because the Legumain deficiency causes the accumulation of pro cathepsins B, H and L, another group of lysosomal cysteine proteases (3). Over-expression of Legumain in tumors is significant for invasion/metastasis (4). Also known as Asparaginyl Endopeptidase, it specifically cleaves peptide bonds with Asn at the P1 position. Nevertheless, it also cleaves peptide bonds with Asp at the P1 position. Auto-activation of pro Legumain involves both types of the cleavage, which result in the removal of the pro peptides in both C- and N-termini (5). In addition, Legumain activates pro MMP-2 and processes bacterial antigens for MHC class II presentation and pro thymosin alpha to thymosin alpha 1 and thymosin alpha 11, two acidic peptides with immunoregulatory properties (6‑8). Human Legumain is synthesized as a 433 amino acid precursor with a signal peptide (residues 1‑17). The pro enzyme (residues 18‑433) was expressed with an N-terminal His tag. This activity of Legumain can be inhibited by recombinant human (rh) Cystatin C and rhCystatin E/M and recombinant mouse Cystatin C (R&D Systems, Catalog # 1196‑PI, 1286-PI and 1238-PI, respectively).
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