All lanes : Anti-Annexin-6/ANXA6 antibody [EPR19517] (ab201023) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ANXA6 knockout HeLa cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : Raji cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 76 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab201023 observed at 75 kDa. Red - loading control ab8245 observed at 36 kDa. 

 ab201023 Anti-Annexin-6/ANXA6 antibody [EPR19536] was shown to specifically react with 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 6/ANXA6 knockout samples were subjected to SDS-PAGE.  ab201023 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.

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling Annexin-6/ANXA6 with ab201023 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasm staining on lymphocytes of 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.

Immunohistochemical analysis of paraffin-embedded Human pancreas tissue labeling Annexin-6/ANXA6 with ab201023 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Membrane staining on 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.

Immunohistochemical analysis of paraffin-embedded Human stomach tissue labeling Annexin-6/ANXA6 with ab201023 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasm staining on stromal cells of Human stomach 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.

All lanes : Anti-Annexin-6/ANXA6 antibody [EPR19517] (ab201023) at 1/5000 dilution

Lane 1 : Human fetal liver lysate
Lane 2 : Human fetal heart lysate
Lane 3 : Human fetal kidney lysate
Lane 4 : Human fetal spleen lysate

Lysates/proteins at 10 µg per lane.

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 why is the actual band size different from the predicted?


Exposure time: 5 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

There was an additional band detected in the heart lysate.

All lanes : Anti-Annexin-6/ANXA6 antibody [EPR19517] (ab201023) at 1/1000 dilution

Lane 1 : A549 (Human lung carcinoma cell line) whole cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 4 : Raji (Human Burkitt's lymphoma cell line) 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

Predicted band size: 76 kDa
Observed band size: 72 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Annexin-6/ANXA6 antibody [EPR19517] (ab201023) at 1/1000 dilution

Lane 1 : Mouse brain lysate at 10 µg
Lane 2 : Mouse heart lysate at 10 µg
Lane 3 : Mouse spleen lysate at 10 µg
Lane 4 : Mouse kidney lysate at 10 µg
Lane 5 : Rat brain lysate at 10 µg
Lane 6 : Rat heart lysate at 10 µg
Lane 7 : Rat kidney lysate at 10 µg
Lane 8 : Rat spleen lysate at 10 µg
Lane 9 : C6 (Rat glial tumor cell line) whole cell lysate at 20 µg
Lane 10 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 µg
Lane 11 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 76 kDa
Observed band size: 72 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1,2 and 3: 15 seconds; Lane 4: 3 minutes; Lane 5,6,7 and 8: 15 seconds; Lane 9 and 10: 5 seconds; Lane 11: 3 minutes.

There was an additional band detected in the heart lysate.

Annexin-6/ANXA6 was immunoprecipitated from 1mg of Human fetal liver whole cell lysate with ab201023 at 1/40 dilution.

Western blot was performed from the immunoprecipitate using ab201023 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: ab201023 IP in Human fetal liver whole cell lysate.

Lane 3: Rabbit IgG,monoclonal [EPR25A] - Isotype Control - (ab172730) instead of ab201023 in Human fetal liver whole cell lysate.

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

Exposure time: 1 second.