United Kingdom (UK)
Recombinant monoclonal antibody to C5aR. Manufactured using AbAb’s Recombinant Platform with variable regions (i.e. specificity) from the hybridoma 32-G1.
UniProt Accession Number of Target Protein: P21730
Alternative Name(s) of Target: CD88; C5a anaphylatoxin chemotactic receptor; C5a anaphylatoxin chemotactic receptor 1; C5a anaphylatoxin chemotactic receptor; C5a-R; C5aR; pC5aR; 32G-1
Immunogen: This antibody was raised by immunizing mice a synthetic C5aR C-terminal phosphopeptide that contained phosphoserine instead of serine at position 334 and also at the neighboring phosphorylation sites Ser-332 and Ser-338.
Specificity: The 32-G1 antibody recognizes C5aR which has its serine 334 phosphorylated. The mAb 32-G1 reacted specifically with a C5aR peptide that contained phosphoserine 334 and also reacted with C5aRs that were isolated from C5a-stimulated, transfected RBL-C5aR cells. It did not recognize a receptor mutant (C5aR-S–) with alanine replacement of all six C-terminal serine phosphorylation sites (Pollok-Kopp et al., 2007). Antibody 32-G1 specifically reacts with C5aR phosphoserine 334, independent of the phosphorylation state of the neighboring serine residues (Pollok-Kopp et al., 2007). Studies with Bt2cAMP-treated U937 cells show that 32-G1 specifically reacts with ligand-activated C5aR at endogenous receptor expression levels but only weakly recognizes C5aR isolated from unstimulated cells (Pollok-Kopp et al., 2007).
Application Notes: The 32-G1 antibody is recommended for the detection and analysis of C5aR (CD88) phosphorylated on Ser-334. For instance, this antibody was successfully used in the identification of C5aR protein in a U937 cell lysate via Western blot (Pollok-Kopp et al., 2007).
Antibody first published in:
Pollok-Kopp et al. Dynamics of protein kinase C-mediated phosphorylation of the complement C5a receptor on serine 334. J Biol Chem. 2007 Feb 16;282(7):4345-53. Epub 2006 Dec 4. PMID:17145764
Note on publication:
This article describes the generation and characterization of the 32-G1 antibody.
Immunofluorescence staining of fixed U937 cells with anti-C5aR antibody 32-G1 (Ab01576) Immunofluorescence analysis of paraformaldehyde fixed U937 cells on Shi-fix™ coverslips stained with the chimeric rabbit IgG version 32-G1 (Ab01576-23.0) at 10 µg/ml for 1h followed by Alexa Fluor® 488 secondary antibody (2 µg/ml), showing membrane staining. The nuclear stain is DAPI (blue). Panels show from left-right, top-bottom Ab01576-23.0, DAPI, merged channels and an isotype control. The isotype control was an unknown specificity antibody (Ab00178-1.1) followed by staining with Alexa Fluor® 488 secondary antibody.
Flow cytometry using the Anti-C5aR antibody 32-G1 (Ab01576). U937 cells were stained with anti-unknown specificity antibody (Ab00178-23.0; isotype control, black line) or the rabbit IgG version of 32-G1 (Ab01576-23.0, blue line) at a dilution of 1:100 for 1h at RT. After washing, the 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.
Flow cytometry using the Anti-C5aR antibody 32-G1 (Ab01576). Paraformaldehyde fixed human peripheral blood monocytes permeabilized with 0.5% Triton were stained with anti-unknown specificity antibody (Ab00178-23.0; isotype control, black line) or the rabbit IgG version of 32-G1 (Ab01576-23.0, blue line) at a dilution of 1:100 for 1h at RT. After washing, the 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.