All lanes : Anti-KIR2DL3 + KIR2DL1 + KIR2DL2 antibody [EPR22541-47] (ab224697) at 1/1000 dilution

Lane 1 : His-tagged human KIR2DL1 recombinant protein (aa1-245)
Lane 2 : His and LIF-tagged human KIR2DL2 recombinant protein (aa26-221)
Lane 3 : His-tagged human KIR2DL3 recombinant protein (aa1-245)
Lane 4 : His-tagged human KIR2DS1 recombinant protein (aa22-221)
Lane 5 : His-tagged human KIR2DS2 recombinant protein (aa22-221)
Lane 6 : His-tagged human KIR2DS3 recombinant protein (aa22-221)
Lane 7 : His-tagged human KIR2DS4 recombinant protein (aa22-221)
Lane 8 : His-tagged human KIR2DS5 recombinant protein (aa22-221) 20 ng

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

Predicted band size: 38 kDa


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded human endometrium tissue labeling KIR2DL2+KIR2DL1+KIR2DL3 with ab224697 at 1/500 dilution (1.13µg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in natural killer (NK) cells in human endometrium (PMID: 7749980). Counterstained with hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NK-92 (human malignant non-Hodgkin's lymphoma natural killer cell) cells labeling KIR2DL2+KIR2DL1+KIR2DL3 with ab224697 at 1/100, 5.7µg/ml dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000, 2µg/ml dilution (Green). Confocal image showing membranous and cytoplasmic staining in NK-92 cells treated with 5-aza-2-deoxycytidine (2µM) for 48 h 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, 2µg/ml dilution.

 

All lanes : Anti-KIR2DL3 + KIR2DL1 + KIR2DL2 antibody [EPR22541-47] (ab224697) at 1/1000 dilution

Lane 1 : Untreated NK-92 (human malignant non-hodgkin's lymphoma natural killer cell) whole cell lysate
Lane 2 : NK-92 treated with 2µM 5-aza-2-deoxycytidine for 48 hours whole cell lysate
Lane 3 : HuT-78 (human sezary syndrome cutaneous t lymphocyte) 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: 38 kDa
Observed band size: 38-60 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

The actual band size higher than predicted is mainly due to glycosylation. The molecular weight observed is consistent with what has been described in the literature  (PMID:11454312 and PMID:12370356).

Blocking/Dilution buffer: 5% NFDM/TBST.

Flow cytometric analysis of human peripheral blood mononuclear cell (PBMC) cells labeling KIR2DL2+KIR2DL1+KIR2DL3 with ab224697 at 1/600 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 rabbit IgG (Left) or ab224697 (Right). Then stained with anti-CD56 conjugated to BV421. Gated on viable cells.

KIR2DL2+KIR2DL1+KIR2DL3 was immunoprecipitated from 0.35 mg NK-92 (Human malignant non-Hodgkin's lymphoma natural killer cell) treated with 2uM 5-aza-2-deoxycytidine for 48h whole cell lysate 10µg with ab224697 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab224697. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: NK-92 (Human malignant non-Hodgkin's lymphoma natural killer cell) treated with 2µM 5-aza-2-deoxycytidine for 48h whole cell lysate 10µg.

Lane 2: ab224697 IP in NK-92 treated with 2µM 5-aza-2-deoxycytidine for 48h whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab224697 in NK-92 treated with 2µM 5-aza-2-deoxycytidine for 48h whole cell lysate.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: 3 mins.