TGF-beta is secreted by cells in the form of an inactive complex. This complex consists of TGF-beta associated non-covalently with a protein designated the latency associated peptide (LAP). TGF-beta and LAP represent components of a pro-peptide that is cleaved in a post-golgi compartment prior to secretion. LAP and TGF-beta each consist of a disulfide-linked homodimer and the association of these two components renders TGF-beta inactive and inaccessible to anti-TGF-beta antibodies.
Applications:
Suitable for use in Flow Cytometry. Other applications not tested.
Recommended Dilution:
Flow Cytometry: 10ul labels (same/less than)1x10e6 cells. Validated using CD4+CD25+ human PBMCs and mouse splenocytes.
Optimal dilutions to be determined by the researcher.
Flow Cytometry Protocol:
1. Cells may be Fc-blocked with 1ug of human or mouse IgG/10e5 cells for 15 minutes at RT. Do not wash excess blocking IgG from this reaction.
2. After blocking add 10ul of L1393-30 to (same/less than)1x10e6 cells and incubate for 30 minutes at RT.
3. Remove unbound antibody by washing the cells twice in Flow Cytometry Staining Buffer (Catalog # F4568-10). Note that whole blood requires a RBC lysis step at this point using Flow Cytometry Human or Mouse Lyse Buffer.
4. Resuspend the cells in Flow Cytometry Staining Buffer for final flow cytometric analysis. As a control for analysis, cells in a separate tube should be treated with PE-labeled mouse IgG1 antibody.
Storage and Stability:
May be stored at 4 degrees C before opening. DO NOT FREEZE! Stable at 4 degrees C as an undiluted liquid. Stable for at least 12 months at 4 degrees C. Freezing R-Phycoerythrin (PE) conjugates will result in a substantial loss of activity. PE conjugates are sensitive to light.
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