Anti-FTO antibody [EPR6895]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FTO antibody [EPR6895] (AB124892)

ab124892, at 1/100 dilution, staining FTO in Paraffin-embedded Human kidney tissue by Immunohistochemistry. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FTO antibody [EPR6895] (AB124892)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human lung carcinoma tissue sections labeling FTO with purified ab124892 at 1 : 100 dilution (1.66 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FTO antibody [EPR6895] (AB124892)

ab124892, at 1/100 dilution, staining FTO in Paraffin-embedded Human Breast tissue by Immunohistochemistry. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FTO antibody [EPR6895] (AB124892)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human breast tissue sections labeling FTO with purified ab124892 at 1 : 100 dilution (1.66 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• WB

Supplier Data

Western blot - Anti-FTO antibody [EPR6895] (AB124892)

Western blot : Anti-TCF12 antibody [7B3] staining at 0.394 ug/ml, shown in black; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab243149 was shown to bind specifically to TCF12. A band was observed at 85 kDa in wild-type Jurkat cell lysates with no signal observed at this size in TCF12 CRISPR-Cas9 edited cell line ab274931 (CRISPR-Cas9 edited cell lysate ab274989). The band observed in the CRISPR-Cas9 edited lysate lane below 85 kDa is likely to represent a truncated form of TCF12. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and TCF12 CRISPR-Cas9 edited Jurkat cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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 before development with Optiblot (ECL reagent ab133456) and imaged with 20 minutes exposure time. Secondary antibodies used were HRP conjugated Goat anti-Rat (H+L) and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-FTO antibody [EPR6895] (ab124892) at 1/1000 dilution

Lane 1:

Wild-type MCF7 cell lysate at 20 µg

Lane 2:

FTO knockout MCF7 cell lysate at 20 µg

Lane 2:

Western blot - Human FTO knockout MCF7 cell line (<a href='/en-us/products/cell-lines/human-fto-knockout-mcf7-cell-line-ab282631'>ab282631</a>)

Lane 3:

HEK-293 cell lysate at 20 µg

Lane 4:

MOLT-4 cell lysate at 20 µg

false

• WB

Lab

Western blot - Anti-FTO antibody [EPR6895] (AB124892)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : FTO knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HEK293 whole cell lysate (20 μg)
Lane 4 : MOLT4 whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab124892 observed at 58 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab124892 was shown to specifically react with FTO in wild-type HAP1 cells. No band was observed when FTO knockout samples were examined. Wild-type and FTO knockout samples were subjected to SDS-PAGE. ab124892 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at a 1/1000 dilution and 1/10,000 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 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-FTO antibody [EPR6895] (ab124892)

Predicted band size: 58 kDa

false

• WB

Lab

Western blot - Anti-FTO antibody [EPR6895] (AB124892)

Western blot : Anti-TCF12 antibody [7B3] staining at 0.394 ug/ml, shown in black; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab243149 was shown to bind specifically to TCF12. A band was observed at 85 kDa in wild-type Jurkat cell lysates with no signal observed at this size in TCF12 CRISPR-Cas9 edited cell line ab274931 (CRISPR-Cas9 edited cell lysate ab274989). The band observed in the CRISPR-Cas9 edited lysate lane below 85 kDa is likely to represent a truncated form of TCF12. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and TCF12 CRISPR-Cas9 edited Jurkat cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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 before development with Optiblot (ECL reagent ab133456) and imaged with 20 minutes exposure time. Secondary antibodies used were HRP conjugated Goat anti-Rat (H+L) and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-FTO antibody [EPR6895] (ab124892) at 1/1000 dilution

Lane 1:

Wild-type MCF7 cell lysate at 20 µg

Lane 2:

FTO knockout MCF7 cell lysate at 20 µg

Lane 2:

Western blot - Human FTO knockout MCF7 cell line (<a href='/en-us/products/cell-lines/human-fto-knockout-mcf7-cell-line-ab282631'>ab282631</a>)

Lane 3:

HEK-293 cell lysate at 20 µg

Lane 4:

MOLT-4 cell lysate at 20 µg

Predicted band size: 58 kDa

Observed band size: 58 kDa

false

• WB

Unknown

Western blot - Anti-FTO antibody [EPR6895] (AB124892)

All lanes:

Western blot - Anti-FTO antibody [EPR6895] (ab124892) at 1/1000 dilution

Lane 1:

293T lysate at 10 µg

Lane 2:

SH-SY5Y lysate at 10 µg

Lane 3:

Caco2 lysate at 10 µg

Lane 4:

BxPC3 lysate at 10 µg

Secondary

All lanes:

Goat anti-Rabbit HRP at 1/2000 dilution

Predicted band size: 58 kDa

false

• WB

Lab

Western blot - Anti-FTO antibody [EPR6895] (AB124892)

All lanes:

Western blot - Anti-FTO antibody [EPR6895] (ab124892) at 1/1000 dilution

Lane 1:

Caco-2 (Human colorectal adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates 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 1/20000 dilution

Predicted band size: 58 kDa

Observed band size: 58 kDa

false

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-FTO antibody [EPR6895] (AB124892)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.