CD99-L2 (CD99 antigen-like 2)

CD99 antigen-like 2 (CD99-L2) is a 45kD type I transmembrane glycoprotein in the CD99 family of molecules (1-3). Mouse CD99-L2 cDNA encodes a 214 amino acid (aa) precursor with a 23 aa predicted signal sequence, a 116 aa extracellular domain (ECD), a 21 aa transmembrane segment, and a 54 aa cytoplasmic region (4). The ECD contains no N-linked glycosylation sites, but O-linked glycosylation is likely (1, 2). A long isoform with a 23 aa insert after aa 43 within the ECD is expressed in human, and probably in mouse (2, 5). Both long and short isoforms may have minor variants missing portions of the N-terminus of the mature protein (6). The ECD of mouse CD99-L2 short isoform shares 85%, 72% and 66% aa identity with rat, human, and bovine CD99-L2, respectively (3, 5). The mouse CD99 and CD99-L2 ECDs share only 31% aa identity, but both contain three conserved acidic motifs and are thought to originate from the same ancestral gene (1, 2). The nearly ubiquitous expression of CD99-L2 is similar to that of CD99. Mouse CD99-L2 shows highest in situ hybridization signals in neurons, cortical thymocytes, ganglia, ovarian granulosa cells, testis, and kidney, and detectable protein levels in lung, thymocytes, mouse leukocytes and vascular endothelial cells (2, 7). CD99-L2 expression on endothelial cells is reported to mediate cell aggregation and neutrophil or monocyte extravasation to inflamed tissue in vivo, while CD99 mediates lymphocyte extravasation as well (3, 7). Applications: Suitable for use in ELISA, Western Blot, Flow Cytometry. Other applications not tested. Recommended Dilution: Western Blot: 0.1-0.2ug/ml with the appropriate secondary reagents to detect mouse CD99-L2. The detection limit for rmCD99-L2 is approximately 2ng/lane under non-reducing and reducing conditions. Flow Cytometry: Dilute this antibody to 25ug/ml and add 10ul of the diluted solution to 1-2.5x10e5 cells in a total reaction volume not exceeding 200ul, using bEnd.3 cells (Schenkel AR, et al., (2007) Cell Commun. Adhes. 14:227). The binding of unlabeled antibodies may be visualized by adding a secondary developing reagent such as anti-goat IgG conjugated to a fluorochrome. Between steps, wash cells in Flow Cytometry Staining Buffer. ELISA: 0.5-1ug/ml with the appropriate secondary reagents to detect mouse CD99-L2. The detection limit for rmCD99-L2 is approximately 0.2ng/well. Optimal dilutions to be determined by the researcher. Endotoxin: (same/less than) 0.25 EU per 1ug of the antibody as determined by the LAL method. Storage and Stability: Lyophilized powder may be stored at -20 degrees C. Stable for 12 months at -20 degrees C. Reconstitute by adding sterile 40-50% glycerol, aliquot and store at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.