Anti-Ferritin heavy chain 抗体 [EPR3004Y]

• WB

Lab

Western blot - Anti-Ferritin heavy chain antibody [EPR3004Y] (AB75973)

False colour image of Western blot : Anti-Ferritin heavy chain antibody [EPR3004Y] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab75973 was shown to bind specifically to Ferritin heavy chain. A band was observed at 24 kDa in wild-type MCF7 cell lysates with no signal observed at this size in FTH1 knockout cell line ab269493 (knockout cell lysate ab269655). To generate this image, wild-type and FTH1 knockout MCF7 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 then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Ferritin heavy chain antibody [EPR3004Y] (ab75973) at 1/1000 dilution

Lane 1:

Wild-type MCF7 cell lysate at 20 µg

Lane 2:

FTH1 knockout MCF7 cell lysate at 20 µg

Lane 3:

Wild-type HEK-293T ab255553 at 20 µg

Lane 4:

FTH1 knockout HEK-293T ab260185 cell lysate at 20 µg

Predicted band size: 21 kDa

Observed band size: 24 kDa

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• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Ferritin heavy chain antibody [EPR3004Y] (AB75973)

Immunofluorescence staining of 4% paraformaldehyde fixed, 0.1% Triton X-100 permeabilized Jurkat cells (human T cell leukemia T lymphocyte from peripheral blood) with ab75973 at a working dilution of 1/100. The secondary antibody, ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed, was used at a dilution of 1/1000. The counterstain, ab195889 anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used at a dilution of 1/200.

Confocal image showing positive staining in Jurkat cells (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Ferritin heavy chain antibody [EPR3004Y] (AB75973)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized SH-SY5Y (Human neuroblastoma epithelial cell) cells labelling Ferritin heavy chain with ab75973 at 1/50 dilution (1μg) (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-Ferritin heavy chain antibody [EPR3004Y] (AB75973)

Lanes 1-3 : Merged signal (red and green). Green - ab75973 observed at 21 kDa. Red - loading control ab8245 observed at 36 kDa.

ab75973 Anti-Ferritin heavy chain antibody [EPR3004Y] was shown to specifically react with Ferritin heavy chain in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266581 (knockout cell lysate ab256924) was used. Wild-type and Ferritin knockout samples were subjected to SDS-PAGE. ab75973 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.

All lanes:

Western blot - Anti-Ferritin heavy chain antibody [EPR3004Y] (ab75973) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

Western blot - Human FTH1 knockout HEK-293T cell lysate (<a href='/products/cell-lysates/human-fth1-knockout-hek-293t-cell-lysate-ab256924'>ab256924</a>) at 20 µg

Lane 3:

SH-SY5Y cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 21 kDa

Observed band size: 21 kDa,36 kDa

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• WB

Unknown

Western blot - Anti-Ferritin heavy chain antibody [EPR3004Y] (AB75973)

Observed MW : 21 kDA

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

All lanes:

Western blot - Anti-Ferritin heavy chain antibody [EPR3004Y] (ab75973) at 1/2000 dilution

Lane 1:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates at 20 µg

Lane 2:

HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates at 20 µg

Lane 3:

C6 (Rat glial tumor glial cell) whole cell lysates at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/20000 dilution

Predicted band size: 21 kDa

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• WB

Lab

Western blot - Anti-Ferritin heavy chain antibody [EPR3004Y] (AB75973)

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

All lanes:

Western blot - Anti-Ferritin heavy chain antibody [EPR3004Y] (ab75973) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) + si -scramble whole cell lysate at 20 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) + si - Ferritin Heavy Chain whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 21 kDa

Observed band size: 21 kDa

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Exposure time: 3s

• WB

Lab

Western blot - Anti-Ferritin heavy chain antibody [EPR3004Y] (AB75973)

Blocking buffer : 5% NFDM/TBST
Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Ferritin heavy chain antibody [EPR3004Y] (ab75973) at 1/10000 dilution

All lanes:

Raw264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 21 kDa

Observed band size: 21 kDa

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• WB

Unknown

Western blot - Anti-Ferritin heavy chain antibody [EPR3004Y] (AB75973)

All lanes:

Western blot - Anti-Ferritin heavy chain antibody [EPR3004Y] (ab75973) at 1/2000 dilution

Lane 1:

Jurkat (Human T cell leukemia cell line from peripheral blood) cell lysate at 10 µg

Lane 2:

SH-SY5Y (Human neuroblastoma cell line from bone marrow) cell lysate at 10 µg

Lane 3:

Human brain lysates at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 21 kDa

Observed band size: 19 kDa,21 kDa

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