Human beta Defensin-2 Recombinant

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Human beta Defensin-2 Recombinant

$70.00$2,700.00

SKU: RKO15263 Categories: , Tag:

accession O15263


Source Optimized DNA sequence encoding Humanbeta Defensin-2mature chain was expressed in Escherichia Coli.
Molecular weight Native humanBD-2 is generated by the proteolytic removal of the signal peptide and propeptide, the molecule has a calculated molecular mass of approximately4 kDa. Recombinant beta Defensin-2 is a monomer protein consisting of42 amino acid residue subunits, and migrates as an approximately4 kDa protein under non-reducing and reducing conditions in SDS-PAGE.
Purity >98%, as determined by SDS-PAGE and HPLC
Biological Activity The ED(50) wasDetermined by its ability to chemoattractimmature human dendritic cells using a concentration range of-50 ng/ml.

Protein Sequence MRVLYLLFSF LFIFLMPLPG VFGGIGDPVT CLKSGAICHP VFCPRRYKQI GTCGLPGTKC CKKP
Endotoxin Endotoxin content was assayed using a LAL gel clot method. Endotoxin level was found to be less than.1 ng/µg(1EU/µg).
Presentation RecombinantHuman betaDefensin-2was lyophilized with no additives
Reconstitution A quick spin of the vial followed by reconstitution inmM Acetic acid to a concentration not less than.1 mg/mL. This solution can then be diluted into other buffers.
Storage The lyophilized protein is stable for at least years from date of receipt at -20° C. Upon reconstitution, this cytokine can be stored in working aliquots at° -° C for one month, or at -20° C for six months, with a carrier protein without detectable loss of activity. Avoid repeated freeze/thaw cycles.
Usage This cytokine product is for research purposes only.It may not be used for therapeutics or diagnostic purposes.


Molecular function Antimicrobial

Methods

Histology & Immunohistochemistry

  • Keratinocyte differentiation was analyzed using primary, antibodies directed against: Elafin (rabbit 92-1), hBD-2 (ab9871, ,), K10 (RKSE60, Eurodiagnostica) and K16 (LL025, Novocastra Laboratories, Newcastle upon Tyne,).
  • Cell division was studied using antibodies against Ki67 (MiB-1, Dako cytomation).
  • To enumerate CD4+ and CD8+ T cells antibodies against CD4 (BC/F6, Santa Cruz Biotechnology, Santa Cruz, CA) and CD8 (144B ) were used.
  • IL-17 production was detected using polyclonal goat IL-17A antibody .
  • To detect Foxp3 expression anti-FoxP3 (PCH101) was used.
  • To detect the presence of human neutrophils and mast cells, antibodies against human neutrophil elastase (NP57) or human mast cell tryptase (AA1) were used.
  • Antibody stainings were visualized using the EnVision+system-HRP kit combined with 3,3′-diaminobenzidine tetrahydrochloride or using that Labeled eptavidin Biotin method (LSAB Kit/AP) combined with either Permanent Red or 5-Bromo-4-Chloro-3-Indolyl Phosphate/Nitro Blue Tetrazolium (BCIP/NBT) .

Chemotaxis of Primary Lymphocytes

  • Peripheral blood lymphocytes were resuspended in RPMI supplemented with 1% FBS at a density of two million cells per mL.
  • Serial dilutions of recombinant human β-defensin 2 (hBD2, , , ) or equivalent vehicle control were prepared in the same media.
  • hBD2 and vehicle dilutions were plated in a ChemoTX plate , alongside media alone controls.
  • The ChemoTX filter was attached to the plate, and 50 µL lymphocyte suspension was applied to the surface of each well.
  • The ChemoTX plate was incubated at 37°C/5% CO2 for 3 hr.
  • To compare migrated cells, media above the filter was removed, and apical surface of filter was washed once in DPBS with 5 mM EDTA, then incubated with the same wash for 30 min at 4°C.
  • This second wash was removed, the ChemoTX plate was centrifuged at 400×g for 5 min, and the filter was removed.
  • Cells in the lower chamber were resuspended in a…
  • Peripheral blood lymphocytes were resuspended in RPMI supplemented with 1% FBS at a density of two million cells per mL.
  • Serial dilutions of recombinant human β-defensin 2 (hBD2, , , ) or equivalent vehicle control were prepared in the same media.
  • hBD2 and vehicle dilutions were plated in a ChemoTX plate , alongside media alone controls.
  • The ChemoTX filter was attached to the plate, and 50 µL lymphocyte suspension was applied to the surface of each well.
  • The ChemoTX plate was incubated at 37°C/5% CO2 for 3 hr.
  • To compare migrated cells, media above the filter was removed, and apical surface of filter was washed once in DPBS with 5 mM EDTA, then incubated with the same wash for 30 min at 4°C.
  • This second wash was removed, the ChemoTX plate was centrifuged at 400×g for 5 min, and the filter was removed.
  • Cells in the lower chamber were resuspended in a volume of 100 µL, and the CytoTox Glo assay was used to compare total cell number according to manufacturer’s instructions.
  • A standard curve of known cell numbers was used to calculate the number of migrated cells from relative light units.

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hBD2 Acid-Urea (AU) Western

hBD2
  • Epithelial cells were harvested and lysed by scraping into 10% acetic acid.
  • These cell lysates were vortexed 30 min at room temperature to extract protein.
  • Soluble extracts were clarified and concentrated, then resolved on an acid-urea polyacrylamide gel electrophoresis (AU-PAGE).
  • A standard of recombinant hBD2 was run alongside cell extracts on each gel.
  • Gels were transferred to PVDF membranes and blotted with a goat polyclonal antibody against hBD2 .

LP01 induces β-defensin expression in primary keratinocytes.

hBD2
  • Quantification of hBD2 and hBD3 mRNA expression in NHEKs treated with LP01 or SECM.