ab214486 staining ANXA1 in HeLa cells. The cells were fixed with 4% paraformaldehyde (10min) permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab214486 at 5ug/ml concentration and ab7291 (Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in pseudo color red).  Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab214486 staining ANXA1 in wild-type Hap1 cells (top panel) and ANXA1 knockout Hap1 cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10min) permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab214486 at 5ug/ml concentration and ab7291 (Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in pseudo color red).  Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: Annexin A1 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)

Lanes 1 - 3: Merged signal (red and green). Green - ab214486 observed at 38 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab214486 was shown to specifically react with Annexin A1 when Annexin A1 knockout samples were used. Wild-type and Annexin A1 knockout samples were subjected to SDS-PAGE. Ab214486 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/2000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling Annexin A1 with ab214486 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. 

Nuclear and cytoplasmic staining on human tonsil tissue is observed [PMID:9720986].

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.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized BxPC-3 (Human pancreas adenocarcinoma cell line) cells labeling Annexin A1 with ab214486 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing membrane and weak cytoplasmic staining on BxPC-3 cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

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

Flow cytometric analysis of 4% paraformaldehyde-fixed NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling Annexin A1 with ab214486 at 1/600 dilution (red) compared with 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 Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

Annexin A1 was immunoprecipitated from 0.35 mg of K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate with ab214486 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab214486 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution

Lane 1: K562 whole cell lysate 10µg (Input).

Lane 2: ab214486 IP in K562 whole cell lysate.

Lane 3: Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab214486 in K562 whole cell lysate.

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

Exposure time: 1 second.

All lanes : Anti-Annexin A1/ANXA1 antibody [EPR19342] (ab214486) at 1/10000 dilution

Lane 1 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate
Lane 2 : BxPC-3 (Human pancreas adenocarcinoma cell line) whole cell lysate
Lane 3 : C2C12 (Mouse myoblast cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 38 kDa
Observed band size: 33,37 kDa why is the actual band size different from the predicted?


Exposure time: 1 second

Blocking/Dilution buffer: 5% NFDM/TBST.

The two band 37kDa is the full length band and the 33kDa supposed to be the cleavage form, this expression pattern observed is consistent with what has been described in the literature (PMID: 25510623 and 20679535).

Immunohistochemical analysis of paraffin-embedded human breast tissue labeling Annexin A1 with ab214486 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear and cytoplasmic staining on human breast tissue is observed [PMID:16949910].

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.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling Annexin A1 with ab214486 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing membrane and cytoplasmic staining on NIH/3T3 cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

Lanes 1-2 : Anti-Annexin A1/ANXA1 antibody [EPR19342] (ab214486) at 1/5000 dilution
Lanes 3-7 : Anti-Annexin A1/ANXA1 antibody [EPR19342] (ab214486) at 1/2000 dilution

Lane 1 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 2 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 3 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate
Lane 4 : Mouse kidney lysate
Lane 5 : Mouse spleen lysate
Lane 6 : Rat kidney lysate
Lane 7 : Rat spleen lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 38 kDa
Observed band size: 33,37 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 3 minutes; Lane 2/6/7: 1 seconds; Lane 3: 15 seconds; Lane 4/5: 30 seconds.

Immunohistochemical analysis of paraffin-embedded human endometrial cancer tissue labeling Annexin A1 with ab214486 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear and cytoplasmic and weak membrane staining on human endometrial cancer tissue 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.

Lanes 1-2 : Anti-Annexin A1/ANXA1 antibody [EPR19342] (ab214486) at 1/5000 dilution
Lane 3 : Anti-Annexin A1/ANXA1 antibody [EPR19342] (ab214486) at 1/20000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : Human fetal kidney lysate
Lane 3 : Human placenta 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: 38 kDa
Observed band size: 33,37 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 3 minutes; Lane 2: 15 seconds; Lane 3: 1 second.

Immunohistochemical analysis of paraffin-embedded human bladder cancer tissue labeling Annexin A1 with ab214486 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear, cytoplasmic and membrane staining on human bladder cancer tissue 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.

Immunohistochemical analysis of paraffin-embedded rat lung tissue labeling Annexin A1 with ab214486 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear and cytoplasmic staining on rat lung tissue is observed [PMID:15133855] [PMID:9720986].

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.

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling Annexin A1 with ab214486 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear and cytoplasmic staining on mouse spleen tissue is observed [PMID:9720986].

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.