Rabbit Anti Human RANKL polyclonal, antigen affinity

///Rabbit Anti Human RANKL polyclonal, antigen affinity

Rabbit Anti Human RANKL polyclonal, antigen affinity

$150.00$190.00


accession O14788


Applications

ELISA:This antibody can be used at 2 μg/mL jointly with biotinylated Human sRANKL antibody to detect Human sRANKL. The detection limit for recombinant Human sRANKL is approximately 0.2 ng/well.

Western Blot:This antibody can be used at 0.2 μg/mL with the appropriate secondary reagents to detect Human sRANKL. The detection limit for recombinant Human sRANKL is approximately 0.2 ng/lane.

Source This antibody was produced in Rabbit immunized with recombinant Human sRANKL.
Species reactivity Human
Purification The specific antibody was purified from Rabbit sera by using immobilized recombinant Human sRANKL affinity chromatography.
Presentation Lyophilized from PBS, pH 7.2.
Storage The lyophilized antibody is stable for at least 1 year from date of receipt at -20° C. Upon reconstitution, this antibody can be stored in working aliquots at 2° - 8° C for one month, or at -20° C for 12 months without detectable loss of activity. Avoid repeated freeze/thaw cycles.
Usage This antibody product is for research purposes only.It may not be used for therapeutics or diagnostic purposes.


Biological Process Differentiation
Molecular function Cytokine
Molecular function Developmental-protein
Molecular function Receptor

Methods

TNFSF11: mRNA transcript variants, genomic loci and sequence conservation A.

a rabbit anti-human RANKL polyclonal antibody
  • Color coded schematics showing the exon structure for the human TNFSF11 mRNA transcripts aligned with the known RANKL protein domains.

Enzyme-linked immunosorbent assay (ELISA)

  • RANKL protein concentration was measured using a sandwich ELISA developed in our laboratory.
  • Recombinant human RANKL was used as a standard.
  • 100 μl/well OPG-FC (&system) was coated onto 96-well microplates at final protein concentrations of 0.25 μg/ml in 50 mM carbonate-bicarbonate buffer, pH 9.6, and incubated overnight at 4°C.
  • The plates were blocked using 5% skim milk, 0.1% Tween-20 in PBS (pH 7.4).
  • 100 μl of supernatant from cultured cells was applied to each well and incubated at room temperature for 2 hours.
  • After washing, the plates were incubated with rabbit anti-hRANKL polyclonal antibody , 250 ng/well for 1 hour at room temperature followed by incubation with an anti-rabbit IgG horseradish peroxidase conjugate .
  • The bound horseradish peroxidase was assayed with 3, 3′, 5, 5′-tetramethylbenzidine (TMB) as substrate, and the reaction was stopped with 2M H2SO4.
  • ODs were determined at 450 nM.
  • The detection limit for this assay was 35 pg/ml.

Sandwich ELISA

  • 96-well maxisorp plates were coated with the capture antibody overnight, then blocked with 5% FCS in PBS for 1 hour, then washed.
  • Then, the protein standard was added, and plates were incubated for 2 hrs at 37°C.
  • Plates were washed and incubated with a biotinylated detection antibody for 1 hour at 37°C.
  • After washing, plates were incubated with HRP-conjugated streptavidin for 30 min at room temperature and washed again.
  • Plates were developed using the tetramethylbenzidine peroxidase substrate system and absorbance was measured at 450-615 nm using an automated plate reader.

Western Blot

  • The Recombinant immunogen was migrated by 12% SDS-PAGE and transferred to a 0.2 um PVDF membrane.
  • After blocking the membrane in blocking buffer (5% milk powder in 20 mM Tris-HCl pH 7.5, 500 mM NaCl, 0.1% (v/v) Tween 20 , the membrane was incubated with primary antibody ( at 4°C overnight.
  • Peroxidase-linked secondary anti-rabbit were used to detect the bound primary antibodies.
  • Enhanced chemiluminescence (ECL) reagents were used to visualize