Mouse MIP-1 alpha (CCL-3) Recombinant

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Mouse MIP-1 alpha (CCL-3) Recombinant

$70.00$2,700.00


accession P10855


Source Optimized DNA sequence encoding Mouse CCL3/Macrophage Inflammatory protein-1 alpha mature chain was expressed in Escherichia Coli.
Molecular weight Native Mouse MIP-1alpha/CCL3 is generated by the proteolytic removal of the signal peptide and propeptide. This molecule has a calculated molecular mass of approximately 8 kDa. Recombinant MIP-1alpha is a monomer protein consisting of 69 amino acid residue subunits,and migrates as an approximately 8 kDa protein under non-reducing andreducing conditions in SDS-PAGE.
Purity >95%, as determined by SDS-PAGE and HPLC
Biological Activity Determined by its ability to chemoattract murine balb/c splenocytes using a concentration range of.0-50.0 ng/ml.

Protein Sequence MKVSTTALAV LLCTMTLCNQ VFSAPYGADT PTACCFSYSR KIPRQFIVDY FETSSLCSQP GVIFLTKRNR QICADSKETW VQEYITDLEL NA
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 MIP-1 alpha was lyophilized from a.2 μm filtered solution in.5% glycine,.5% sucrose,.01% Tween80, mM Glutamic acid, pH.5.
Reconstitution A quick spin of the vial followed by reconstitution in distilled water 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.


Interactor E9M5R0
Biological Process Chemotaxis
Biological Process Inflammatory-response
Molecular function Cytokine

Methods

Ccr1 ligands are induced after Candida infection and are chemotactic for kidney neutrophils ex vivo.

recombinant mouse CCL3
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Binding and in vitro activity of murine 18V4F hybridoma antibody.

CCL3
  • Directly coated chemokines were used here for direct comparisons, however in other experiments CCL3 showed a significantly enhanced signal when biotinylated and coated on streptavidin plates.

Binding and in vitro activity of murine 18V4F hybridoma antibody.

CCL3
  • Directly coated chemokines were used here for direct comparisons, however in other experiments CCL3 showed a significantly enhanced signal when biotinylated and coated on streptavidin plates.

Flow cytometric analyses

  • PBMC were cultured in the presence of adefovir, tenofovir, and PMEO-DAPym and incubated at 37°C in a humidified, 5% CO2-controlled atmosphere.
  • The expression of cellular activation markers was measured after 3 days of culture.
  • Briefly, after washing the cells with PBS containing 2% FCS, we incubated them with PerCP-conjugated anti-CD4 mAb (clone SK3) in combination with PE-conjugated anti-CD25, anti-CD69, or anti-HLA-DR mAbs for 30 min at room temperature.
  • For aspecific background staining, cells were stained in parallel with Simultest Control IgG γ1/γ2a and PerCP-conjugated mouse IgG1.
  • Finally, the cells were washed with PBS, fixed with 1% formaldehyde solution, and analyzed with a FACSCalibur ; data were acquired with CellQuest software and further analyzed with the FLOWJO software .
  • The expression of the chemokine receptors was measured after 24 h, and PBMC were incubated with PerCP-conjugated anti-CD4 mAb (clone SK3) in combination with APC-conjugated anti-CXCR4 mAb (clone 12G5) and PE-conjugated anti-CCR5 (clone 2D7) or…
  • PBMC were cultured in the presence of adefovir, tenofovir, and PMEO-DAPym and incubated at 37°C in a humidified, 5% CO2-controlled atmosphere.
  • The expression of cellular activation markers was measured after 3 days of culture.
  • Briefly, after washing the cells with PBS containing 2% FCS, we incubated them with PerCP-conjugated anti-CD4 mAb (clone SK3) in combination with PE-conjugated anti-CD25, anti-CD69, or anti-HLA-DR mAbs for 30 min at room temperature.
  • For aspecific background staining, cells were stained in parallel with Simultest Control IgG γ1/γ2a and PerCP-conjugated mouse IgG1.
  • Finally, the cells were washed with PBS, fixed with 1% formaldehyde solution, and analyzed with a FACSCalibur ; data were acquired with CellQuest software and further analyzed with the FLOWJO software .
  • The expression of the chemokine receptors was measured after 24 h, and PBMC were incubated with PerCP-conjugated anti-CD4 mAb (clone SK3) in combination with APC-conjugated anti-CXCR4 mAb (clone 12G5) and PE-conjugated anti-CCR5 (clone 2D7) or anti-CXCR3 (clone 1C6).
  • To evaluate the biological effects of the β-chemokines produced by drug-treated PBMC, freshly isolated PBMC were incubated for 1 h at 37°C with supernatants collected from PBMC cultures treated with compounds for 24 h. Then, cells were incubated with PerCP-conjugated anti-CD4 mAb (clone SK3) in combination with PE-conjugated anti-CCR5 mAb (clone 2D7) for 30 min at room temperature.
  • As a control, cells were also incubated for 1 h at 37°C with the LD78β isoform of MIP-1 alpha , which is described as potently downregulating the CCR5 receptor

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In vivo neutrophil recruitment into the cremaster muscle

  • Male C57Bl/6 mice of similar age were injected with an intrascrotal injection of 200 µl with either saline (sterile NaCl) or COAM (0.2 mg) 3 or 24 h prior to experiments.
  • At the time for experiment, mice were anaesthetized by spontaneous inhalation of isoflurane gas via an isoflurane pump ( 400 , ’s , , ) through a breathing mask containing a mixture of air and oxygen (total oxygen 40%) and ∼2.4% isoflurane.
  • The animals were placed on a water-heated operating table to maintain body temperature at ∼37°C.
  • The depth of anesthesia was controlled by regularly monitoring peripheral reflexes.
  • The cremaster muscle was prepared as previously described 2) was analyzed.
  • The cremaster muscles were saved for further analyses.