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78144
LILRB1/CD85j (D4L8L) Rabbit mAb
Primary Antibodies
Monoclonal Antibody

LILRB1/CD85j (D4L8L) Rabbit mAb #78144

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  1. WB
Western Blotting Image 1 - LILRB1/CD85j (D4L8L) Rabbit mAb

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 Blotting Image 2 - LILRB1/CD85j (D4L8L) Rabbit mAb

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 Blotting Image 3 - LILRB1/CD85j (D4L8L) Rabbit mAb

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).

To Purchase # 78144S
Product # Size Price
78144S
100 µl $ 268

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Supporting Data

REACTIVITY H
SENSITIVITY Endogenous
MW (kDa) 110
Source/Isotype Rabbit IgG

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

Product Usage Information

Application Dilution
Western Blotting 1:1000

Storage

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.

Protocol

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Western Blotting Protocol

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 product webpage for recommended antibody dilution.

A. Solutions and Reagents

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.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, mix.
  3. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH2O.
  4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH2O, mix.
  5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH2O, mix.
  6. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
  7. Nonfat Dry Milk: (#9999).
  8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
  9. Wash Buffer: (#9997) 1X TBST.
  10. Bovine Serum Albumin (BSA): (#9998).
  11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
  12. Biotinylated Protein Ladder Detection Pack: (#7727).
  13. Prestained Protein Marker, Broad Range (11-190 kDa): (#13953).
  14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  15. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
  16. Detection Reagent: SignalFire™ ECL Reagent (#6883).

B. Protein Blotting

A general protocol for sample preparation.

  1. Treat cells by adding fresh media containing regulator for desired time.
  2. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
  3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
  4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
  5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
  6. Microcentrifuge for 5 min.
  7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

    NOTE: Loading of prestained molecular weight markers (#13953, 5 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.

  8. Electrotransfer to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.

I. Membrane Blocking

  1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
  2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
  3. Wash three times for 5 min each with 15 ml of TBST.

II. Primary Antibody Incubation

  1. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product webpage) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
  2. Wash three times for 5 min each with 15 ml of TBST.
  3. Incubate membrane with Anti-rabbit IgG, HRP-linked Antibody (#7074 at 1:2000) and anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
  4. Wash three times for 5 min each with 15 ml of TBST.
  5. Proceed with detection (Section D).

D. Detection of Proteins

Directions for Use:

  1. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
  2. Prepare 1X SignalFire™ ECL Reagent (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
  3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated exposure to skin.

posted June 2005

revised June 2020

Protocol Id: 10

Specificity / Sensitivity

LILRB1/CD85j (D4L8L) Rabbit mAb recognizes endogenous levels of total LILRB1/CD85J protein. This antibody does not cross-react with LILRB2 protein.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg370 of human LILRB1/CD85j protein.

Background

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).

  1. Allan, D.S. et al. (2000) Immunobiology 202, 34-41.
  2. Roberti, M.P. et al. (2015) Eur J Immunol 45, 1560-9.
  3. Colonna, M. et al. (1997) J Exp Med 186, 1809-18.
  4. Samaridis, J. and Colonna, M. (1997) Eur J Immunol 27, 660-5.
  5. Borges, L. et al. (1997) J Immunol 159, 5192-6.
  6. Shiroishi, M. et al. (2003) Proc Natl Acad Sci U S A 100, 8856-61.
  7. Naji, A. et al. (2014) J Immunol 192, 1536-46.

Pathways & Proteins

Explore pathways + proteins related to this product.

For Research Use Only. Not For Use In Diagnostic Procedures.

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