Our Abpromise guarantee covers the use of ab10858 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Notes
Inhibition Assay
  • Application notesELISA: Use at an assay dependent dilution. The detection limit for recombinant human BMP5 is approximately 0.16 ng/well.
    IHC-Fr: Use at a concentration of 1 - 3 µg/ml.
    IHC-P: Use at a concentration of 5 - 10 µg/ml.
    Inhib: Use at an assay dependent dilution.
    WB: Use at an assay dependent dilution. Predicted molecular weight: 20 kDa.

    Cells may be fixed for 20 minutes at room temperature with freshlyprepared 1 to 2 % paraformaldehyde/PBS (pH 7.4). Three to five washes of the cells in PBS (15 minutes each) is usually required after fixation and before the addition of the primary antibody (anti-BMP5). Labeling may be obtained by incubating the cells overnight at 4 °C with at least 1 to 3 µg/ml anti-BMP5 antibody followed by the appropriate secondary reagents.

    For tissue immunohistochemistry, a working concentration of at least 5 to 10 µg/ml antibody is recommended to detect human BMP5 on cryostat sections 5 to 15 µm thick. Dissected tissues should be fixed by vascular perfusion with 4 % paraformaldehyde/ PBS (pH 7.4) and followed by perfusion with a 10 % sucrose solution in 0.1 M phosphate buffer (pH 7.2). Note: prolonged fixation in 4 % paraformaldehyde can diminish the labeling and staining intensity of BMP5. Cryostat sections from 5 to 15 µm are incubated with the primary antibody (anti-BMP5) at concentrations of at least 5 to 10 µg/ml. Note: on freefloating sections, anti-BMP5 should be diluted to at least 0.3 to 1 µg/ml.

    The labeling detection system on cells and tissues can be done by either fluorescent or non-fluorescent enzymatic assays. Due to autofluorescence in neuronal tissues, the use of fluorescence detectors such as FITC or Cy3 is not recommended unless the autofluorescence is quenched (example: treating tissues after IHC staining with 1% Sudan Black in 70 % alcohol). Non-fluorescent enzymatic staining using DAB, AEC, or immunogold-silver can be used.

    Anti-human BMP5 has the ability to neutralize the biological activity of recombinant human BMP5 on MC3T3-E1 cells. Recombinant human BMP5 is added to various concentrations of the antibody for 1 hour at 37 °C in a 96 well microplate. Following this pre-incubation, MC3T3-E1 cells are added to the mixture. The assay mixture in a total volume of 100 µl, containing antibody at concentrations of 0.01 µg/ml to 100 µg/ml, recombinant human BMP5 at 1.5 µg/ml, L-ascorbic acid at 50 µg/ml, and cells at 5 x 104 cells/ml, is incubated at 37 °C for 4 days in a humidified CO2 incubator. At the end of the incubation, alkaline phosphatase activity in cell lysate is measured.

    The Neutralization Dose50 (ND50) for anti-human BMP5 is approximately 3 to 10 µg/ml in the presence of 1.5 µg/ml of recombinant human BMP5, using the MC3T3-E1 cell line. The ND50 is the concentration of antibody required to yield one-half maximal inhibition of the cytokine activity on a responsive cell line, when the cytokine is present at a concentration just high enough to elicit a maximum response. The exact concentration of antibody required to neutralize human BMP5 activity is dependent on the cytokine concentration, cell type, growth conditions, and the type of activity studied.

    The detection limit for recombinant human BMP5 is approximately 5 ng/lane under non-reducing and reducing conditions. Recombinant BMP5, a disulfide-linked homodimeric protein, consists of two 167 amino acid residue subunits with a calculated molecular mass of approximately 18 kDa. Due to glycosylation, the protein migrates as a doublet of 20 kDa and 25 kDa under reducing conditions in SDS-PAGE. BMP5 is synthesized as a large precursor protein that is cleaved at the dibasic cleavage site (RXXR) to release the carboxy-terminal domain.

    Not tested in other applications.
    Optimal dilutions/concentrations should be determined by the end user.
  • Target

    References for Anti-BMP5 antibody (ab10858)

    ab10858 has not yet been referenced specifically in any publications.

    Product Wall

    Thank you for your enquiry. This is a polyclonal antibody that was raised to the full-length protein. Thius, the antiserum will consist of a mixed population of IgGs, each of which will recognise a different epitope along the target protein. I h...

    Read More