重组Anti-Apolipoprotein E抗体[EPR19392]

• WB

Lab

Western blot - Anti-Apolipoprotein E antibody [EPR19392] (AB183597)

False colour image of Western blot : Anti-Apolipoprotein E antibody [EPR19392] staining at 1/2000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab183597 was shown to bind specifically to Apolipoprotein E. A band was observed at 34 kDa in wild-type HepG2 cell lysates with no signal observed at this size in APOE knockout cell line. To generate this image, wild-type and APOE knockout HepG2 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % milk in TBS-0.1 % Tween^® 20 (TBS-T) 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:

Western blot - Anti-Apolipoprotein E antibody [EPR19392] (ab183597) at 1/2000 dilution

Lane 1:

Wild-type HepG2 cell lysate at 20 µg

Lane 2:

APOE knockout HepG2 cell lysate at 20 µg

Lane 2:

Western blot - Human APOE knockout Hep G2 cell line (<a href='/products/cell-lines/human-apoe-knockout-hep-g2-cell-line-ab280875'>ab280875</a>)

Lane 3:

Human Liver cell lysate at 20 µg

Lane 4:

Human Kidney cell lysate at 20 µg

Predicted band size: 36 kDa

Observed band size: 34 kDa

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• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Apolipoprotein E antibody [EPR19392] (AB183597)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed HepG2 (Human liver hepatocellular carcinoma cell line) cells labeling Apolipoprotein E with ab183597 at 1/70 dilution (red) compared with a Rabbit IgG,monoclonal- Isotype control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (Alexa Fluor^® 488) at 1/500 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Apolipoprotein E antibody [EPR19392] (AB183597)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling Apolipoprotein E with ab183597 at 1/2000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on macrophages of Human tonsil is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Apolipoprotein E antibody [EPR19392] (AB183597)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling Apolipoprotein E with ab183597 at 1/2000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on hepatocytes of Human liver is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Apolipoprotein E antibody [EPR19392] (AB183597)

Immunofluorescent analysis of 100% methanol-fixed HepG2 (Human liver hepatocellular carcinoma cell line) cells labeling Apolipoprotein E with ab183597 at 1/500 dilution followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HepG2 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG (AlexaFluor^®594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows :
-ve control 1 : ab183597 at 1/500 dilution followed by ab150120 at 1/1000 dilution.
-ve control 2 : ab7291 at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Apolipoprotein E antibody [EPR19392] (AB183597)

Immunohistochemical analysis of formalin fixed paraffin embedded human liver labelling Apolipoprotein E with ab183597 at a concentration of 0.1 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH 8.5 for 32mins.

ab183597 anti- Apolipoprotein E [EPR19392] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Apolipoprotein E antibody [EPR19392] (AB183597)

Immunohistochemical analysis of paraffin-embedded Rat cerebral cortex tissue labeling Apolipoprotein E with ab183597 at 1/4000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on astrocytes of rat cerebral cortex is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Apolipoprotein E antibody [EPR19392] (AB183597)

Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling Apolipoprotein E with ab183597 at 1/4000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on hepatocytes of rat liver is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Apolipoprotein E antibody [EPR19392] (AB183597)

Immunohistochemical analysis of paraffin-embedded Mouse thalamus tissue labeling Apolipoprotein E with ab183597 at 1/4000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on astrocytes of mouse thalamus is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Apolipoprotein E antibody [EPR19392] (AB183597)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling Apolipoprotein E with ab183597 at 1/4000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on hepatocytes of mouse liver and plasma was also stained. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

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Immunoprecipitation - Anti-Apolipoprotein E antibody [EPR19392] (AB183597)

Apolipoprotein E was immunoprecipitated from 1mg of Mouse plasma with ab183597 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab183597 at 1/2000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : Mouse plasma, 10μg (Input).

Lane 2 : ab183597 IP in Mouse plasma.

Lane 3 : Rabbit IgG,monoclonal[EPR25A] - Isotype Control (ab172730) instead of ab183597 in Mouse plasma.

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

Exposure time : 10 seconds.

All lanes:

Immunoprecipitation - Anti-Apolipoprotein E antibody [EPR19392] (ab183597)

Predicted band size: 36 kDa

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• WB

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Western blot - Anti-Apolipoprotein E antibody [EPR19392] (AB183597)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Apolipoprotein E antibody [EPR19392] (ab183597) at 1/2000 dilution

Lane 1:

Human fetal liver lysate at 10 µg

Lane 2:

Human fetal kidney lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 36 kDa

Observed band size: 36 kDa

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Exposure time: 30s

• WB

Supplier Data

Western blot - Anti-Apolipoprotein E antibody [EPR19392] (AB183597)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 : 3 minutes; Lane 2 and 3 : 30 seconds; Lane 4, 5 and 6 : 5 seconds.

All lanes:

Western blot - Anti-Apolipoprotein E antibody [EPR19392] (ab183597) at 1/2000 dilution

Lane 1:

Rat liver lysate at 20 µg

Lane 2:

Mouse liver lysate at 20 µg

Lane 3:

HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

Lane 4:

Human plasma at 20 µg

Lane 5:

Mouse plasma at 20 µg

Lane 6:

Rat plasma at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 36 kDa

Observed band size: 36 kDa

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• WB

Supplier Data

Western blot - Anti-Apolipoprotein E antibody [EPR19392] (AB183597)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 and 2 : 5 seconds; Lane 3 : 10 seconds; Lane 4 : 30 seconds.

All lanes:

Western blot - Anti-Apolipoprotein E antibody [EPR19392] (ab183597) at 1/2000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Mouse heart lysate at 10 µg

Lane 3:

Rat brain lysate at 10 µg

Lane 4:

Rat kidney lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 36 kDa

Observed band size: 36 kDa

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• WB

Lab

Western blot - Anti-Apolipoprotein E antibody [EPR19392] (AB183597)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000000 dilution.

All lanes:

Western blot - Anti-Apolipoprotein E antibody [EPR19392] (ab183597) at 1/1000 dilution

Lane 1:

Wild-type mouse cerebral cortex tissue lysate at 20 µg

Lane 2:

APOE knockout mouse cerebral cortex tissue lysate at 20 µg

Lane 3:

Wild-type mouse liver tissue lysate at 20 µg

Lane 4:

APOE knockout mouse liver tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

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Exposure time: 5s