Anti-Annexin-6/ANXA6 antibody [EPR19536]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Annexin-6/ANXA6 antibody [EPR19536] (AB201024)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Raji (Human Burkitt's lymphoma cell line) cells labeling Annexin-6/ANXA6 with ab201024 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 Raji cells.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows : -

-ve control 1 : ab201024 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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Annexin-6/ANXA6 antibody [EPR19536] (AB201024)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed HepG2 (Human liver hepatocellular carcinoma cell line) cells labeling Annexin-6/ANXA6 with ab201024 at 1/600 dilution (red) compared with a a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (Alexa Fluorr^® 488) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Annexin-6/ANXA6 antibody [EPR19536] (AB201024)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HepG2 (Human liver hepatocellular carcinoma cell line) cells labeling Annexin-6/ANXA6 with ab201024 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 cells.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows : -

-ve control 1 : ab201024 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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Annexin-6/ANXA6 antibody [EPR19536] (AB201024)

Immunohistochemical analysis of paraffin-embedded Human pancreas tissue labeling Annexin-6/ANXA6 with ab201024 at 1/8000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasm and membrane staining on the Human pancreas islet is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is 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-Annexin-6/ANXA6 antibody [EPR19536] (AB201024)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling Annexin-6/ANXA6 with ab201024 at 1/8000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasm staining on Human tonsil is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is 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-Annexin-6/ANXA6 antibody [EPR19536] (AB201024)

Annexin-6/ANXA6 was immunoprecipitated from 0.35mg of Human fetal liver whole cell lysate with ab201024 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab201024 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : Human fetal liver whole cell lysate,10μg (Input).

Lane 2 : ab201024 IP in Human fetal liver whole cell lysate.

Lane 3 : Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab201024 in Human fetal liver whole cell lysate.

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

Exposure time : 3 seconds.

All lanes:

Immunoprecipitation - Anti-Annexin-6/ANXA6 antibody [EPR19536] (ab201024)

Predicted band size: 76 kDa

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• WB

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Western blot - Anti-Annexin-6/ANXA6 antibody [EPR19536] (AB201024)

Blocking/Dilution buffer : 5% NFDM/TBST.

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

All lanes:

Western blot - Anti-Annexin-6/ANXA6 antibody [EPR19536] (ab201024) at 1/5000 dilution

Lane 1:

Human skeletal muscle lysate at 20 µg

Lane 2:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Lane 3:

Raji (Human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

Lane 4:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 5:

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

Secondary

All lanes:

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

Predicted band size: 76 kDa

Observed band size: 72 kDa

false

• WB

Lab

Western blot - Anti-Annexin-6/ANXA6 antibody [EPR19536] (AB201024)

Lanes 1-4 : Merged signal (red and green). Green - ab201024 observed at 75 kDa. Red - loading control ab8245 observed at 36 kDa.

ab201024 Anti-Annexin-6/ANXA6 antibody [EPR19536] was shown to specifically react with Annexin-6/ANXA6 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265677 (knockout cell lysate ab257351) was used. Wild-type and Annexin-6/ANXA6 knockout samples were subjected to SDS-PAGE. ab201024 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Annexin-6/ANXA6 antibody [EPR19536] (ab201024) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

ANXA6 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human ANXA6 (Annexin-6) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-anxa6-annexin-6-knockout-hela-cell-line-ab265677'>ab265677</a>)

Lane 3:

Jurkat cell lysate at 20 µg

Lane 4:

Raji cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 76 kDa

Observed band size: 75 kDa

false

• WB

Unknown

Western blot - Anti-Annexin-6/ANXA6 antibody [EPR19536] (AB201024)

Blocking/Dilution buffer : 5% NFDM/TBST.

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

All lanes:

Western blot - Anti-Annexin-6/ANXA6 antibody [EPR19536] (ab201024) at 1/1000 dilution

Lane 1:

Human fetal liver lysate at 10 µg

Lane 2:

Human fetal heart lysate at 10 µg

Lane 3:

Human fetal kidney lysate at 10 µg

Lane 4:

Human spleen 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: 76 kDa

Observed band size: 72 kDa

false