Anti-Ferritin heavy chain antibody [EPR3004Y] - BSA and Azide free

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Ferritin heavy chain antibody [EPR3004Y] - BSA and Azide free (AB271854)

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).

This data was developed using the same antibody clone in a different buffer formulation (ab75973).

• WB

Lab

Western blot - Anti-Ferritin heavy chain antibody [EPR3004Y] - BSA and Azide free (AB271854)

This data was developed using the same antibody clone in a different buffer formulation (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] (<a href='/en-us/products/primary-antibodies/ferritin-heavy-chain-antibody-epr3004y-ab75973'>ab75973</a>) 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

false

• WB

Lab

Western blot - Anti-Ferritin heavy chain antibody [EPR3004Y] - BSA and Azide free (AB271854)

This data was developed using ab75973, the same antibody clone in a different buffer formulation.

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

All lanes:

Western blot - Anti-Ferritin heavy chain antibody [EPR3004Y] (<a href='/en-us/products/primary-antibodies/ferritin-heavy-chain-antibody-epr3004y-ab75973'>ab75973</a>) 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='/en-us/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

false

Exposure time: 3s

• WB

Lab

Western blot - Anti-Ferritin heavy chain antibody [EPR3004Y] - BSA and Azide free (AB271854)

This data was developed using the same antibody clone in a different buffer formulation (ab75973). Blocking buffer : 5% NFDM/TBST
Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Ferritin heavy chain antibody [EPR3004Y] (<a href='/en-us/products/primary-antibodies/ferritin-heavy-chain-antibody-epr3004y-ab75973'>ab75973</a>) 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='/en-us/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

false