Anti-FTO antibody [EPR24440-12]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-FTO antibody [EPR24440-12] (AB280081)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 cells labelling FTO with ab280081 at 1/50 (11.98 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing nuclear and weak cytoplasmic staining in NIH/3T3 cell line is observed.

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-FTO antibody [EPR24440-12] (AB280081)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labelling FTO with ab280081 at 1/500 dilution (0.1ug)/ (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• WB

Lab

Western blot - Anti-FTO antibody [EPR24440-12] (AB280081)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

Exposure time : Lanes 1-2 : 15 seconds; Lanes 3-5 : 6 seconds; Lanes : 6-7 : 15 seconds.

All lanes:

Western blot - Anti-FTO antibody [EPR24440-12] (ab280081) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 2:

C2C12 (mouse myoblasts myoblast) whole cell lysate at 20 µg

Lane 3:

HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 4:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

HAP1 (human chronic myelogenous leukemia near-haploid cell line) whole cell lysate at 20 µg

Lane 6:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Lane 7:

L6 (rat skeletal muscle myoblast) 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 1/50000 dilution

Predicted band size: 58 kDa

Observed band size: 60 kDa

false

• WB

Lab

Western blot - Anti-FTO antibody [EPR24440-12] (AB280081)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

Exposure time : 3 minutes.

All lanes:

Western blot - Anti-FTO antibody [EPR24440-12] (ab280081) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Mouse heart tissue lysate at 20 µg

Lane 3:

Rat brain tissue 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 1/50000 dilution

Predicted band size: 58 kDa

Observed band size: 60 kDa

false

• WB

Lab

Western blot - Anti-FTO antibody [EPR24440-12] (AB280081)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

Exposure time : Lanes 1-2 : 15 seconds;Lane 3 : 48 seconds.

All lanes:

Western blot - Anti-FTO antibody [EPR24440-12] (ab280081) at 1/1000 dilution

Lane 1:

Human lung tissue lysate 20 at 20 µg

Lane 2:

Human kidney tissue lysate at 20 µg

Lane 3:

Human skeletal muscle tissue lysate at 20 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 58 kDa

Observed band size: 60 kDa

false

• WB

Lab

Western blot - Anti-FTO antibody [EPR24440-12] (AB280081)

Blocking and diluting buffer and concentration : 3% NFDM/TBST

Lanes 1-3 : Merged signal (red and green). Green - ab280081 observed at 60 kDa. Red - loading control ab8245 observed at 36 kDa.

ab280081 Anti-FTO antibody [EPR24440-12] was shown to specifically react with FTO in wild-type HAP1 cells. Loss of signal was observed when the knockout cell line was used. Wild-type and FTO knockout samples were subjected to SDS-PAGE. ab280081 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4℃ overnight 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 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-FTO antibody [EPR24440-12] (ab280081) at 1/1000 dilution

Lane 1:

Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate at 20 µg

Lane 2:

FTO knockout HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate at 20 µg

Lane 3:

HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Predicted band size: 58 kDa

Observed band size: 60 kDa

false