Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of HEK-293 tissue labelling (A) - HEK-293 transfected with NKG2A expression vector containing a myc-His-tag®, (B) - HEK-293 transfected with NKG2C expression vector containing a myc-His-tag®, (C) - HEK-293 transfected with NKG2E expression vector containing a myc-His-tag® whole cell pellets and (D) -  HEK-293 transfected with an empty vector with ab260035 at 1/200 dilution. Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins, Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Positive staining on (A) HEK-293 transfected with NKG2A expression vector containing a myc-His-tag®, whole cell pellet. No staining on (B) HEK-293 transfected with NKG2C expression vector containing a myc-His-tag®, whole cell pellet and (C) HEK-293 transfected with NKG2E expression vector containing a myc-His-tag®, whole cell pellet. and (D) HEK-293 transfected with an empty vector.
The section was incubated with ab260035 for30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized Jurkat (Human T cell leukemia T lymphocyte, Left) / NK-92 (Human malignant non-Hodgkin's lymphoma natural killer cell, Right) cells labelling NKG2A with ab260035 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled 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.

Negative control: Jurkat (PMID: 15699146).

Immunohistochemical analysis of paraffin-embedded Human NK cell lymphoma tissue labeling NKG2A with ab260035 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human NK cell lymphoma (PMID: 11222501).The section was incubated with ab260035 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

All lanes : Anti-NKG2A antibody [EPR23737-127] (ab260035) at 1/1000 dilution

Lane 1 : NK-92 ( human malignant non-hodgkins lymphoma natural killer cell) whole cell lysate
Lane 2 : Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/50000 dilution

Predicted band size: 26 kDa
Observed band size: 34,35,37-45 kDa why is the actual band size different from the predicted?

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

Multiple bands are likely due to isoforms and glycosylation. The molecular profile/weight observed is consistent with what has been described in the literature (PMID: 9034158).

Negative control: Jurkat (PMID: 15699146).

Exposure time: 70 seconds.

NKG2A was immunoprecipitated from 0.35 mg NK-92 (human malignant non-Hodgkin's lymphoma natural killer cell) whole cell lysate with ab260035 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using 260035 at 1/1000 dilution. 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) whole cell lysate 10 ug

Lane 2: 260035 IP in NK-92 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab260035 in NK-92 whole cell lysate

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

Exposure time: 58 seconds.

Multiple bands are likely due to isoforms and glycosylation. The molecular profile/weight observed is consistent with what has been described in the literature (PMID: 9034158).

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NK-92 cells labelling NKG2A with ab260035 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing membranous and cytoplasmic staining in NK-92 cell line. Negative control: Jurkat (PMID: 15699146). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - 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: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 (2 ug/ml) dilution.

All lanes : Anti-NKG2A antibody [EPR23737-127] (ab260035) at 1/5000 dilution

Lanes 1 & 3 & 5 : HEK-293 (human embryonic kidney) transfected with an empty vector (vector control), containing a myc-His-tag®, whole cell lysate
Lane 2 : HEK-293 transfected with NKG2A (WT) expression vector containing a myc-His-tag®, whole cell lysate
Lane 4 : HEK-293 transfected with NKG2C (WT) expression vector containing a myc-His-tag®, whole cell lysate
Lane 6 : HEK-293 transfected with NKG2E (WT) expression vector containing a myc-His-tag®, whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/50000 dilution

Predicted band size: 26 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?

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

Exposure time: 3 seconds.

Flow cytometric analysis of 2% paraformaldehyde fixed, 0.1% Tween-20 permeabilized Human peripheral blood mononuclear cell (PBMC) cells labelling NKG2A with ab260035 at 1/500 dilution (0.1ug)/ Right  compared with a Rabbit monoclonal IgG (ab172730) isotype control (Left). 

A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Cells were stained with anti-CD56 conjugated to PE, then fixed with 2% PFA followed by intracellularly stained with rabbit IgG (Left) or ab260035 (Right).

Immunohistochemical analysis of paraffin-embedded Human gastric cancer tissue labeling NKG2A with ab260035 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human gastric cancer (PMID: 11222501).The section was incubated with ab260035 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling NKG2A with ab260035 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human tonsil (PMID: 11222501). The section was incubated with ab260035 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.