Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NK-92 (human peripheral blood malignant non-Hodgkin's lymphoma cell line) cells (+/- treatment with 5-aza-2-deoxycytidine (2µM) for 48h) labeling KIR2DL1 + KIR2DL2 with ab252936 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous staining in NK-92 cells treated with 5-aza-2-deoxycytidine(2µM) for 48h. The nuclear counterstain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

KIR2DL1 + KIR2DL2 were immunoprecipitated from 0.35 mg of NK-92 (human peripheral blood malignant non-Hodgkin's lymphoma cell line) (treated with 2μM 5-aza-2-deoxycytidine for 48h) whole cell lysate with ab252936 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab252936 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.

Lane 1: NK-92 (treated as above) whole cell lysate 10 μg (Input). 
Lane 2: ab252936 IP in NK-92 (treated as above) whole cell lysate. 
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab252936

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 30 seconds.

 

Immunohistochemical analysis of paraffin-embedded human endometrium tissue labeling KIR2DL1 + KIR2DL2 with a252936 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining in natural killer (NK) cells in human endometrium (PMID: 7749980) is observed. Counterstained with hematoxylin.

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

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Flow cytometric analysis of human PBMCs (peripheral blood mononuclear cells) labeling KIR2DL1 + KIR2DL2 with ab252936 at 1/50 dilution (Right) compared with Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (Left).

Goat Anti-Rabbit IgG Fc (Alexa Fluor^® 488) preadsorbed (ab150097), at 1/5000 dilution was used as the secondary antibody.

Cells were stained with rabbit IgG (Left) or ab252936 (Right). Then stained with anti-CD56 conjugated to BV421.

Gated on viable cells.

 

All lanes : Anti-KIR2DL1 + KIR2DL2 antibody [EPR22492-92] (ab252936) at 1/1000 dilution

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


Exposure time: 26 seconds

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-KIR2DL1 + KIR2DL2 antibody [EPR22492-92] (ab252936) at 1/1000 dilution

Lane 1 : Untreated NK-92 (human peripheral blood malignant non-Hodgkin's lymphoma cell line) whole cell lysate
Lane 2 : NK-92 (treated with 2µM 5-aza-2-deoxycytidine for 48) 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: 39 kDa
Observed band size: 39-60 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking and dilution buffer: 5% NFDM/TBST.

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).

This blot was developed using a higher sensitivity ECL substrate.