United Kingdom (UK)
Recombinant monoclonal antibody to Podoplanin (MAP tag). Manufactured using AbAb’s Recombinant Platform with variable regions (i.e. specificity) from the hybridoma PMab-1.
UniProt Accession Number of Target Protein: Q62011
Alternative Name(s) of Target: PDPN; Aggrus; Glycoprotein 38; Gp38; OTS-8; Ots8; PA2.26 antigen; T1-alpha; T1A; Transmembrane glycoprotein E11
Immunogen: This antibody was raised by immunising rats with 14-residue synthetic peptide mpp3851, which corresponds to amino acids 38-51 (GDGMVPPGIEDKIT) of the platelet-aggregation-stimulating (PLAG) domain of mouse podoplanin. Spleen cells were then harvested, and fused to P3U1 cells to generate stable hybridomas.
Specificity: This antibody is specific for amino acids 38-51 (GDGMVPPGIEDKIT) of the platelet-aggregation-stimulating (PLAG) domain of mouse podoplanin (PDPN). This antibody, therefore, possesses high affinity and specificity for the MAP epitope tag (GDGMVPPGIEDK), which is derived from the PLAG domain of murine podoplanin. PDPN is a type I transmembrane protein that interacts with the platelet receptor C-type lectin-like receptor-2 (CLEC-2). PDPN is expressed by a wide variety of cells, including podocytes, lymphatic endothelial cells and type I alveolar cells. PDPN is also upregulated on certain cancer cells, where it mediates tumour-induced platelet aggregation by binding to CLEC-2 on platelets. Chihara (2018) associated Podoplanin with a co-inhibitory receptor in T-cell activation along with PD-1, TIM-3, LAG-3 and TIGIT.
Application Notes: This antibody binds to the PLAG domain of murine PDPN and is, therefore, able to block its interaction with C-type lectin-like receptor-2 (CLEC-2) (Yamada, 2017). This has a number of potential therapeutic applications, including the suppression of lymphangiogenesis and inflammatory macrophage infiltration at the site of wound healing in mouse corneal suture and ear section models (Maruyama et al, 2014), as well as suppression of transplant rejection. This antibody possesses high affinity and specificity for the MAP epitope tag (GDGMVPPGIEDK), and can, therefore, be used to detect and purify tagged proteins in a range of assay formats (Fujii et al, 2016). PMab-1 performs well in immunohistochemical analysis, and has been used to stain the mouse melanoma B16-F10 and mouse podoplanin-expressing CHO transfectants (Kaji et al, 2012). The use of this antibody in immunostaining of somatic tissues, including mouse lung lymphatic vessels, kidney glomeruli, pulmonary alveoli, pulmonary pleura and salivary gland myoepithelial cells, has been successfully performed on both paraffin-embedded sections and frozen sections (Kaji et al, 2012). The specificity of this antibody has been confirmed in Western blot analysis (Kaji et al, 2012), while Western blot analysis has also revealed that activation of LPS-induced inflammatory pathways, including the MAPK and NF-κB pathways, is affected by PMab-1 (Maruyama et al, 2014). The mouse–rat chimeric antibody mPMab-1 is derived from PMab-1 (Yamada et al, 2017).
Antibody first published in:
Kaji et al Immunohistochemical Examination of Novel Rat Monoclonal Antibodies against Mouse and Human Podoplanin Acta Histochem. Cytochem. 45 (4): 227–237, 2012. doi:10.1267/ahc.12008 PMID:23012488
Note on publication:
Describes the original generation and characterisation of this antibody.
Western Blot using anti-Podoplanin (MAP tag) antibody PMab-1 (Ab00875-23.0) Mouse thymus (A), mouse brain (B), NIH3T3 cells (C) and mouse kidney (D) lysates (35µg protein in RIPA buffer) were resolved on a 10% SDS PAGE gel and blots probed with the chimeric rabbit IgG version of 1B4 (Ab01258-23.0) at 0.01 µg/ml, 0.1 µg/ml, 0.001 µg/ml and 0.003 µg/ml, respectively, before detection using an anti-rabbit secondary antibody. A primary incubation of 1h was used and protein was detected by chemiluminescence.
Flow-cytometry using the anti-Podoplanin (MAP tag) antibody PMab-1 (Ab00875) NIH3T3 cells were stained with anti-Fluorescein IgG antibody (4-4-20; isotype control, black line) or the rabbit IgG-chimeric version of PMab-1 (Ab00875-23.0, blue line) at a dilution of 1:100 for 1h at RT. After washing, bound antibody was detected using a goat anti-rabbit IgG AlexaFluor® 488 antibody at a dilution of 1:1000 and cells analyzed using a FACSCanto flow-cytometer.
ELISA using PMab-1 (Ab00875) and Multi Tag protein 2. Binding curves of the mouse IgG1 chimeric version of the anti-MAP antibody PMab-1 (Ab00875-1.1; ed line) and isotype control (Ab00102-1.1, anti-fluorescein; orange line) to an ELISA plate coated with Multi Tag protein 2 (Pr00565, Absolute Antibody) at a concentration of 5 ug/ml. Ab02007 antibody was titrated on a 3-fold serial dilution starting at 10,000 ng/mL. Anti-mouse IgG was used as secondary antibody for detection at a dilution of 1:4,000.
Western Blot using Anti-Podoplanin (MAP tag) antibody PMab-1 (Ab00875). Podoplanin (MAP tag) expressing plasmid transfected (A) and non-transfected (B) HEK293 cells lysate (3µg protein in RIPA buffer) were resolved on a SDS PAGE gel and blots were probed with the chimeric rabbit version of PMab-1 (Ab00875-23.0) at 0.00001µg/ml before detection using an anti-rabbit secondary antibody. A primary incubation of 1h was used and protein was detected by chemiluminescence.
Immunofluorescence staining of HEK293 cells with Anti-Podoplanin (MAP tag) (Ab00875) PMab-1 Immunofluorescence analysis of paraformaldehyde fixed HEK293 cells transfected with Podoplanin (MAP tag) expressing plasmid (A) and non-transfected HEK293 cells (B), permeabilized with 0.15% Triton stained with the chimeric rabbit IgG version of PMab-1 (Ab00875-23.0) (1:200 dilution) for 1h followed by Alexa Fluor® 488 secondary antibody (1:1000 dilution), showing membrane and cytoplasmic staining. The nuclear stain is DAPI (blue).