Western blot analysis of extracts from Daudi cells, untreated (-) or treated with PNGase F (+), using LILRB1/CD85j (D4L8L) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from various cell lines using LILRB1/CD85j (D4L8L) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). As expected, LILRB1/CD85j protein is not detected in A-431 cells.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human LILRB1 protein (hLILRB1-Myc/DDK; +) or Myc/DDK-tagged full-length human LILRB2 protein (hLILRB2-Myc/DDK; +), using LILRB1/CD85j (D4L8L) Rabbit mAb (upper), DYKDDDDK Tag Antibody #2368 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
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.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
LILRB1/CD85j (D4L8L) Rabbit mAb recognizes endogenous levels of total LILRB1/CD85J protein. This antibody does not cross-react with LILRB2 protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg370 of human LILRB1/CD85j protein.
The leukocyte Ig-like receptor subfamily B (LILRB) are type-I transmembrane glycoproteins containing ligand binding extracellular IgG-like domains and immunoreceptor tyrosine-based inhibition motifs (ITIMS) within the cytoplasmic domain, which recruit SHP protein tyrosine phosphatases, leading to transduction of signals that inhibit immune cell activation. Encoded within a region of chromosome 19 known as the leukocyte receptor complex, the LILRB subfamily of inhibitory receptors consists of LILRB1 to LILRB5, also referred to as CD85J, CD85D, CD85A, CD85K, and CD85C, respectively (1). There is mounting evidence that LILRBs function, in part, as a novel class of immune checkpoint receptors and support tumor growth through the transmission of inhibitory signals upon engagement of ligands expressed on tumor cells (2).
LILRB1 (CD85j/ILT2/LIR1) is expressed across multiple hematopoietic cell lineages, such as B-cells, NK cells, monocytes, and T-cells (3,4). Like other members of the LILRB subfamily, LILRB1 contains multiple extracellular IgG-like domains and intracellular ITIM motifs (5). Research studies have demonstrated that LILRB1 interacts with multiple HLA class I ligands, such as HLA-G (6). Cross-linking of LILRB1 upon ligand engagement has been shown to activate immunosuppressive signaling cascades, which in B-cells, suppresses their activation and ability to produce antibodies (7). In NK and T cell lineages, research studies have shown that LILRB1 transduces signaling to inhibit cytolytic activity (3).
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