All lanes : Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572) at 1/500 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CTNNB1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 85 kDa
Observed band size: 86 kDa why is the actual band size different from the predicted?

This data was developed using the same antibody clone in a different buffer formulation (ab32572).

  Lanes 1- 2: Merged signal (red and green). Green - ab32572 observed at 86 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab32572 was shown to react with beta Catenin in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255352 (knockout cell lysate ab263756) was used. Wild-type HeLa and CTNNB1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab32572 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 500 Dilution and a 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.

ab32572 staining in CTNNB1 (beta Catenin) wild-type HAP1 cells (top panel) and in CTNNB1 (β-catenin) knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), 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 ab32572 at 1/250 dilution and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody at 2 μg/ml (shown in green). Nuclear DNA was labeled in blue with DAPI.

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32572).

Different expression level of beta Catenin in Human HCTs (hepatocellular carcinoma tissues) and PLTs (para-cancerous liver tissues).

The HCTs, PLTs were paraffin-embedded and cut into sections with 5 μm-thickness for hematoxylin-eosin and immunohistochemistry (IHC) analysis. ab32572 was used at a dilution of 1:400. The second antibody was a biotinylated IgG to incubate 40 minutes at 37°C. Finally, the tissue slices were visualized by the 3, 3-diaminobenzidine solution and counterstained with hematoxylin. Substitution of the primary antibody with phosphate-buffered saline was served as a control for IHC.

The beta Catenin with negative, weak, moderate and strong staining activity was respectively detected in HCTs (E-H) and PLTs (M-P). Section E shown above, for full image please see original paper.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32572).

All lanes : Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572) at 1/5000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : CTNNB1 (ß-catenin) knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : A431 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 85 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab32572 observed at 90 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32572 was shown to specifically react with CTNNB1 (β-catenin) in wild type HAP1 cells. No band was observed when CTNNB1 (β-catenin) knockout samples were used. Wild-type and CTNNB1 (β-catenin) knockout samples were subjected to SDS-PAGE. ab32572 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at a 1/5000 dilution and 1/10000 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 1/20000 dilution for 1 hour at room temperature before imaging.

This data was developed using the same antibody in a different buffer formulation (ab32572).

Chromatin was prepared from HCT 116 cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30 mins and then formaldehyde for 10 min.

The ChIP was performed with 25 µg of chromatin, 5 µg of ab32572 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 20 µl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci)

Primers and probes are from paper PMID: 28625518

*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

ab32572 staining beta Catenin in the bEnd.5 murine cell line by ICC/IF (Immunocytochemistry/immunofluorescence).

Cells were fixed with paraformaldehyde, permeabilized with 0.1% Triton in PBS and blocked with 10% serum for 30 minutes at 22°C. Samples were incubated with primary antibody (1/300) for 16 hours at 4°C. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32572).

ab32572 staining beta Catenin in dog colon tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections).

Tissue was fixed with formaldehyde and blocked with 10% serum for 30 minutes at 25°C; antigen retrieval was by heat mediation in 10 mM citrate buffer, pH6. Samples were incubated with primary antibody (1/250 in PBS with 1x casein) for 90 minutes at 25°C. A biotin-conjugated Goat anti-rabbit IgG polyclonal (1/200) was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32572).

ab32572 at 1/200 staining mouse small intestine tissue sections by IHC-P.

The tissue was formaldehyde fixed and a heat mediated antigen retrieval step was performed before incubation with the primary antibody. An HRP conjugated goat anti-rabbit antibody was used as the secondary.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32572).

ab32572 staining human renal carcinoma tissue sections by IHC-P. 

Sections were formaldehyde fixed and subjected to heat mediated antigen retrieval in citrate buffer (pH 6) prior to blocking with 1% milk for 45 minutes at 22°C.  The primary antibody was diluted 1/200 and incubated with the sample for 1 hour at 22°C.  An HRP conjugated goat anti-rabbit antibody, diluted 1/400, was used as the secondary.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32572).

Clone E247 (ab196204) has been successfully conjugated by Abcam. This image was generated using Anti-beta Catenin antibody [E247] (Alexa Fluor® 488). Please refer to ab194118 for protocol details.

ab194118 staining β-catenin in wild-type HAP1 cells (top panel) and β-catenin knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), 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 ab194118 at 1/500 dilution (shown in green) and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

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

Clone E247 (ab196204) has been successfully conjugated by Abcam. This image was generated using Anti-beta Catenin antibody [E247] (Alexa Fluor® 647). Please refer to ab194119 for protocol details.

ab194119 staining β-catenin in wild-type HAP1 cells (top panel) and β-catenin knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), 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 ab194119 at 1/500 dilution (shown in red) and ab195887 at 1/250 dilution (shown in green) overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

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

ab32572 staining beta Catenin in SW480 (Human colorectal adenocarcinoma cell line) cells treated with BIO (ab120891), by ICC/IF. Increase of beta Catenin expression correlates with increased concentration of BIO, as described in literature.
The cells were incubated at 37°C for 48h in media containing different concentrations of ab120891 (BIO) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab32572 (1/200) dilution was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A Goat Anti-Rabbit IgG H&L (DyLight^® 488) preadsorbed (ab96899) secondary antibody at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32572).

ab32572 showing positive staining in human cervical carcinoma tissue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32572).

ab32572 showing positive staining in human breast carcinoma tissue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32572).

ab32572 showing positive staining in human lung adenocarcinoma tissue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32572).

ab32572 showing positive staining in human papillary carcinoma of thyroid gland tissue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32572).

ab32572 showing positive staining in human kidney carcinoma tissue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32572).

ab32572 staining beta Catenin in SK-N-SH (Human neuroblastoma cell line) cells treated with olanzapine (ab120736), by ICC/IF.

Increase in expression of beta Catenin correlates with increased concentration of olanzapine, as described in literature.
The cells were incubated at 37°C for 24h in media containing different concentrations of ab120736 (olanzapine) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab32572 (1/200 dilution) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A Goat Anti-Rabbit IgG H&L (DyLight^® 488) preadsorbed (ab96899) secondary antibody at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32572).