All lanes : Anti-LILRB1 antibody [EPR22861-6] (ab238145) at 1/1000 dilution

Lane 1 : IM-9 (human multiple myeloma b lymphoblast) whole cell lysate
Lane 2 : K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 71 kDa
Observed band size: 90-110 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Diluting buffer and concentration: 5% NFDM/TBST.

The molecular weight observed is consistent with what has been described in the literature (PMID: 22844324).

Negative control: K-562 (PMID: 22844324).

LILRB1 was immunoprecipitated from 0.35 mg IM-9 (Human multiple myeloma B Lymphoblast) whole cell lysate with ab238145 at 1/30 dilution (2 μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab238145 at a 1/1000 dilution (0.48 μg/ml). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used as the secondary antibody at 1/5000 dilution.

Lane 1: IM-9 (Human multiple myeloma B Lymphoblast) whole cell lysate 10μg
Lane 2: ab238145 IP in IM-9 whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab238145 in IM-9 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 30 seconds.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized human PBMC (human primary peripheral blood mononuclear cell) cells labeling LILRB1 with ab238145 at 1/50 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing membranous staining in subsets of human PBMC is observed. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)  was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Flow cytometric analysis of 2% paraformaldehyde fixed 0.1% Tween-20 permeabilized HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) transfected with myc-tagged LILRB2 expression vector (Left) / HEK-293T transfected with myc-tagged LILRB1 expression vector (Right) cells labeling LILRB1 with ab238145 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. Cells were surface stained with rabbit IgG (black) or ab238145 (red). Then fixed with 2% PFA followed by intracellularly stained with anti-myc tag conjugated to Alexa Fluor^® 647. Gated on myc+ population.

Flow cytometric analysis of human peripheral blood mononuclear cell (PBMC) cells labeling LILRB1 with ab238145 at 1/500 dilution (0.1μg)/ Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150097) at 1/5000 dilution was used as the secondary antibody. Cells were stained with anti-CD11b conjugated to BV421. Then stained with rabbit IgG (Left) or ab238145 (Right). Gated on viable cells.