Human beta Defensin-2 Recombinant

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Human beta Defensin-2 Recombinant

$70.00 – $2,700.00

SKU: RKO15263 Categories: PDGF family, PDGF family Tag: Defensin

Description

Methods (4)


accession	O15263

Source	Optimized DNA sequence encoding Human beta Defensin-2 mature chain was expressed in Escherichia Coli.
Molecular weight	Native human BD-2 is generated by the proteolytic removal of the signal peptide and propeptide, the molecule has a calculated molecular mass of approximately 4 kDa. Recombinant beta Defensin-2 is a monomer protein consisting of 42 amino acid residue subunits, and migrates as an approximately 4 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) was determined by its ability to chemoattract immature human dendritic cells using a concentration range of 0-50 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	Recombinant Human beta Defensin-2 was lyophilized with no additives
Reconstitution	A quick spin of the vial followed by reconstitution in 10mM Acetic acid to a concentration not less than 0.1 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. Read more
hBD2 Acid-Urea (AU) Western 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. Quantification of hBD2 and hBD3 mRNA expression in NHEKs treated with LP01 or SECM.