Anti-HLA E 抗体 [EPR25300-104] (BSA and Azide free) (ab300554)

All lanes : Anti-HLA E antibody [EPR25300-104] (ab300553) at 1/1000 dilution

Lane 1 : Wild-type A549 (human lung carcinoma epithelial cell), whole cell lysate
Lane 2 : HLA-E knockout A549 whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (ab216773) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (ab216776) at 1/20000 dilution

Observed band size: 40 kDa why is the actual band size different from the predicted?

This data was developed using ab300553, the same antibody clone in a different buffer formulation. 

Blocking and diluting buffer and concentration: Lysates at 20 µg per lane. 

Performed under reducing conditions.

False colour image of Western blot: Anti-HLA E antibody [EPR25300-104] (ab300553) staining at 1/1000 dilution, shown in green; Mouse anti-Tubulin antibody [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red.

In Western blot, ab300553 was shown to bind specifically to HLA E. A band was observed at 40kDa in wild-type A549 cell lysates with no signal observed at this size in HLA E knockout cell line ab267080 (knockout cell lysate ab258452). To generate this image, wild-type and HLA Eknockout A549 cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in in Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes : Anti-HLA E antibody [EPR25300-104] (ab300553) at 1/1000 dilution

Lane 1 : Human liver tissue lysate
Lane 2 : Human placenta tissue lysate
Lane 3 : HL-60 (human Acute Promyelocytic Leukemia promyeloblast), whole cell lysate
Lane 4 : K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate
Lane 5 : Daudi (human Burkitts lymphoma lymphoblast), whole cell lysate
Lane 6 : THP-1 (human monocytic leukemia monocyte), 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

Observed band size: 40 kDa why is the actual band size different from the predicted?

This data was developed using ab300553, the same antibody clone in a different buffer formulation. 

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

The 1, 4, 5, and 6 lanes of this blot were developed using a high sensitivity ECL substrate.

Exposure time: Lane 1-3: 103 seconds Lane 4-6: 3 minutes

This data was developed using ab300553, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human spleen tissue labeling HLA E with ab300553 at 1/500 dilution (0.106 µg/mL) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection kit). Positive staining on human spleen. The section was incubated with ab300553 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody, followed by a ready to use secondary antibody LeicaDS9800 (Bond™ Polymer Refine Detection kit).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

This data was developed using ab300553, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling HLA E with ab300553 at 1/500 dilution (0.106 µg/mL) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection kit). Positive staining on endothelial cells and Kupffer cells of human liver. The section was incubated with ab300553 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody, followed by a ready to use secondary antibody LeicaDS9800 (Bond™ Polymer Refine Detection kit).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

This data was developed using ab300553, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Wild-type A549 (Human lung carcinoma epithelial cell (cell pellet)) labeling HLA E with ab300553 at 1/100 (5.3 µg/mL) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection kit). Positive staining on (A) wild-type A549 cell pellet, no staining on (B) HLA-E knockout A549 (ab267080) cell pellet. The section was incubated with ab300553 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

This data was developed using ab300553, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human diffuse large tissue labeling HLA E with ab300553 at 1/500 dilution (0.106 µg/mL) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection kit). Positive staining on human diffuse large B cell lymphoma. The section was incubated with ab300553 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody, followed by a ready to use secondary antibody LeicaDS9800 (Bond™ Polymer Refine Detection kit).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

This data was developed using ab300553, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 80% Methanol-fixed, 0.1% TritonX-100 permeabilized THP-1 (human monocytic leukemia monocyte) cells labeling HLA E with ab300553 at 1/50 (10.6 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 (2 µg/ml) dilution (Green). Confocal image showing membrane and cytoplasmic staining on THP-1 cell line is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (2.5µg/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 (2 µg/ml) dilution.

This data was developed using ab300553, the same antibody clone in a different buffer formulation.

HLA E was immunoprecipitated from 0.35 mg THP-1 (human monocytic leukemia monocyte), whole cell lysate 10 µg with ab300553 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab300553 at ID285 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: THP-1 (human monocytic leukemia monocyte), whole cell lysate 10 µg

Lane 2: ab300553 IP in THP-1 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300553 in THP-1 whole cell lysate

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

Exposure time: 3 minutes