Western blot analysis of extracts from HDLM-2 and Jurkat cells using LAG3 (D2G4O™) XP® Rabbit mAb (Biotinylated) (upper) and β-Actin (13E5) Rabbit mAb #4970 (lower).
This Cell Signaling Technology antibody is conjugated to biotin under optimal conditions. The biotinylated antibody is expected to exhibit the same species cross-reactivity as the unconjugated LAG3 (D2G4O™) XP® Rabbit mAb #15372.
Supplied in 136 mM NaCl, 2.6 mM KCI, 12 mM sodium phosphate (pH 7.4) dibasic, 2 mg/ml BSA, and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
Do not add Anti-biotin, HRP-linked Antibody for detection of biotinylated protein markers. There is no need. The Streptavidin-HRP will also visualize the biotinylated markers.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 265
LAG3 (D2G4O™) XP® Rabbit mAb (Biotinylated) recognizes endogenous levels of total LAG3 protein.
Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the amino terminus of human LAG3 protein.
Lymphocyte activation gene 3 (LAG-3, CD223) is an immune checkpoint control protein that negatively regulates T cells and immune responses. A CD4-like member of the Ig superfamily, LAG3 contains an extracellular IgV and three IgC domains, a transmembrane domain, and a short cytoplasmic region (1). LAG3 is primarily expressed by activated CD4+ T cells, CD8+ T cells, Tregs and NK cells, where it's activated by MHC Class II molecules, its only known ligand. While it was initially shown to activate Treg cells (2), LAG3 can also inhibit CD8+ T cells (3,4). LAG3 is often co-expressed with PD-1 on the surface of tumor infiltrating lymphocytes, where the two proteins act independently to contribute to tumor-mediated immune suppression (4,5). Blockade of LAG3 is a promising strategy for neoplastic intervention (6).
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D2G4O is a trademark of Cell Signaling Technology. Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
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