Anti-Apolipoprotein E4 antibody [EPR24181-64] (ab279714) at 1/1000 dilution + Human plasma at 5 µl

Secondary
Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 36 kDa
Observed band size: 36 kDa

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

Exposure time:  3 minutes

All lanes : Anti-Apolipoprotein E4 antibody [EPR24181-64] (ab279714) at 1/1000 dilution

Lane 1 : HEK-293T (human embryonic kidney) transfected with an empty vector (vector control), containing a myc-His-tag®, whole cell lysate
Lane 2 : HEK-293T transfected with Apolipoprotein E2 (R176C) expression vector containing a myc-His-tag®, whole cell lysate
Lane 3 : HEK-293T transfected with Apolipoprotein E3 expression vector containing a myc-His-tag®, whole cell lysate
Lane 4 : HEK-293T transfected with Apolipoprotein E4 (C130R) 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 (HRP) (ab97051) at 1/50000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 36 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?

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

Exposure time:  3 minutes

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling Apolipoprotein E4 with ab279714 at 1/1000 dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human kidney. (PMID:29692990 ) The section was incubated with ab279714 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

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

Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labeling Apolipoprotein E4 with ab279714 at 1/1000 dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human breast cancer. The section was incubated with ab279714 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

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

Immunohistochemical analysis of paraffin-embedded Human placenta (plasma) tissue labeling Apolipoprotein E4 with ab279714 at 1/1000 dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on the plasma in human placenta. (PMID: 26828652) The section was incubated with ab279714 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

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

Immunohistochemical analysis of paraffin-embedded sections Panel A and D: HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) transfected with APOE4 (C130R) expression vector containing a myc-His-tag®
Panel B: HEK-293T transfected with APOE2 (R176C) expression vector containing a myc-His-tag®. Panel C: HEK-293T transfected with APOE3 expression vector containing a myc-His-tag® labeling Apolipoprotein E4 with ab279714 at 1/1000 dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on (A) HEK-293T transfected with an APOE4 (C130R) expression vector is observed. Almost no staining on (B) HEK-293T transfected with an APOE2 (R176C) expression vector and (C) HEK-293T transfected with APOE3 expression vector. (D) Secondary antibody only control for (A). The section was incubated with ab279714 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

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

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T cells labelling Apolipoprotein E4 with ab279714 at 1/1000 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing strong cytoplasmic staining in 293T cells transfected with APOE4-Myc plasmid is observed. Myc-Tag Mouse mAb (Alexa Fluor® 647) antibody was used to counterstain transfected proteins 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 dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T cells labelling Apolipoprotein E4 with ab279714 at 1/500 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution. Negative control: Confocal image showing no staining in 293T cells transfected with APOE2-Myc or APOE3-Myc plasmid.. Myc-Tag Mouse mAb (Alexa Fluor® 647) antibody was used to counterstain transfected proteins 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 dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized 293T (Human embryonic kidney epithelial cell) transfected with myc-tagged Apolipoprotein E4 construct cells labeling Apolipoprotein E4 with ab279714 at 1/500 dilution (Right panel) compared with Rabbit monoclonal IgG Isotype Control (ab172730) (Left panel). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized 293T (Human embryonic kidney epithelial cell) transfected with myc-tagged Apolipoprotein E2 construct (Left)/ 293T transfected with myc tagged Apolipoprotein E3 construct (Right) labeling Apolipoprotein E4 with ab279714 at 1/500 dilution.  Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Apolipoprotein E4 was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney) transfected with Apolipoprotein E4 (C130R) expression vector containing a myc-His-tag® whole cell lysate 10 μg with ab279714 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab279714 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1:HEK-293T (human embryonic kidney) transfected with Apolipoprotein E4 (C130R) expression vector containing a myc-His-tag® whole cell lysate 10 μg

Lane 2: ab279746 IP in HEK-293T (human embryonic kidney) transfected with Apolipoprotein E4 (C130R) expression vector containing a myc-His-tag® whole cell lysate 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab279714 in HEK-293T transfected with Apolipoprotein E4 (C130R) expression vector containing a myc-His-tag® whole cell lysate

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

Exposure time: 41 seconds