Western blot analysis of human Monocyte Chemotactic Protein-4 (MCP-4)/CCL13 was performed by loading 2.5ug of recombinant human MCP-4 protein per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with StartingBlock (TBS) Blocking Buffer (Product # 37542) for at least 1 hour. The membrane was probed with an MCP4 monoclonal antibody (Product # M809) at a concentration of 2ug/ml overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween-20, and probed with a goat anti-mouse IgG-HRP secondary antibody (Product # 31430) at a dilution of 1:5000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080).
|Tested species reactivity||Human|
|Host / Isotype||Mouse / IgG1|
|Immunogen||Recombinant CCL13 protein|
|Storage buffer||PBS with 1mg/ml BSA, 30% glycerol|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|ELISA (ELISA)||2 µg/ml|
|Western Blot (WB)||2 µg/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
There is one major form (long chain) and two minor forms (short chain and medium chain) of MCP-4 produced by differential signal peptide cleavage. Thus, the resulting MW of secreted MCP-4 ranges between ~8-9kD. Western blot analysis of recombinant MCP-4/CCL13 protein detects an ~17kD band, likely corresponding to a dimer of MCP-4.
The M809 MCP-4 antibody (clone 3G4) has successfully been paired as the coating antibody in a sandwich ELISA with detection antibody M810 (biotinylated conjugate of clone 8C12). The M809 MCP-4 antibody (biotinylated conjugate of clone 3G4) has also been used successfully in a sandwich ELISA as a detection antibody when paired with M810 (clone 8C12) as the coating antibody.
Cytokines are a family of secreted proteins involved in immunoregulatory and inflammatory processes. The gene encoding MCP-4/CCL13 is one of several Cys-Cys (CC) cytokine genes clustered on the q-arm of chromosome 17. The CC cytokines are proteins characterized by two adjacent cysteines. The cytokine encoded by this gene displays chemotactic activity for monocytes, lymphocytes, basophils and eosinophils, but not neutrophils. MCP-4/CCL13 signals through CCR2B and CCR3 receptors, and plays a role in the accumulation of leukocytes at both sides of allergic and non-allergic inflammation. It be also involved in the recruitment of monocytes into the arterial wall during the disease process of atherosclerosis, and may also play a role in monocyte attraction in tissues chronically exposed to exogenous pathogens.
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