hMCP-1-induced migration of THP-1 cells was assessed. THP-1 cells were incubated in a 96-well transwell plate with increasing concentrations of hMCP-1 in the bottom chamber. After 2 hr, the number of THP-1 cells that migrated to the bottom chamber of the transwell was quantified by measuring DNA content using a fluorescent dye.
The purity of recombinant hMCP-1 was determined by SDS-PAGE of 6 µg reduced (+) and nonreduced (-) recombinant hMCP-1 and staining overnight with Coomassie Blue.
With carrier: Lyophilized from a 0.22 μm filtered solution of hMCP-1 in 20 mM Tris, pH 7.2 containing 20 μg BSA per 1 μg hMCP-1. Carrier free: Lyophilized from a 0.22 μm filtered solution of hMCP-1 in 20 mM Tris, pH 7.2.
Stable in lyophilized state at 4°C for 1 year after receipt. Sterile stock solutions reconstituted with carrier protein are stable at 4°C for 2 months and at -20°C for 6 months. Avoid repeated freeze-thaw cycles. Maintain sterility. Storage at -20°C should be in a manual defrost freezer.
>95% as determined by SDS-PAGE of 6 μg reduced (+) and nonreduced (-) recombinant hMCP-1. All lots are greater than 95% pure.
Molecular Characterization: Recombinant hMCP-1 has a calculated MW of 8,685. DTT-reduced protein migrates as a 10 kDa polypeptide. The nonreduced protein migrates at 12 kDa. The expected amino-terminal QPDAI of recombinant hMCP-1 was verified by amino acid sequencing.
The activity of hMCP-1 was determined using a THP-1 cell migration assay. The ED50 of each lot is between 1-11 ng/ml.
Less than 0.01 ng endotoxin/1 μg hMCP-1.
Recombinant Human MCP-1 (hMCP-1) Gln24-Thr99 (Accession #NP_002973) was expressed in E.coli at Cell Signaling Technology.
MCP-1 (CCL2) is the first member of the C-C family of chemokines to be identified (1). C-C chemokines are characterized by two adjacent cysteine residues within the polypeptide, which form an intra-molecular disulfide bond. MCP-1 is a potent chemotactic factor for monocytes/macrophages, T cells and a subset of NK cells (1-4). The MCP-1 receptor, CCR2, is expressed as two splice isoforms, CCR2A and CCR2B, of which CCR2B is the predominant form (1). MCP-1 is secreted by adipocytes and appears to be one of many links between obesity, inflammation, and diabetes (1). MCP-1/CCR2 signaling appears to play a key role in γδ effector T cells recruitment and anti-tumor responses in a murine B16 melanoma model (2). Conversely, CCL2 expression is upregulated in many types of cancer and has been implicated in promoting tumor cell survival, proliferation, and tumor associated inflammation (4).
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