Anti-Heme Oxygenase 1 抗体 [EPR1390Y]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Heme Oxygenase 1 antibody [EPR1390Y] (AB68477)

Immunocytochemistry/Immunofluorescence analysis of HepG2 (Human liver hepatocellular carcinoma cell line) cells labeling Heme Oxygenase 1 with purified ab68477 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000. DAPI was used as a nuclear counterstain.

Negative control 1 : PBS only.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Heme Oxygenase 1 antibody [EPR1390Y] (AB68477)

Intracellular Flow Cytometry analysis of A549 (human lung carcinoma) cells labeling with purified ab68477 at 1/200 dilution (1ug/ml) (Red). Cells were fixed with4% paraformaldehydeand permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (ab150077) (1/2000 dilution) was used as the secondary antibody.Rabbit monoclonal IgG (Black) (ab172730) was used as a isotype control.Cell without incubation with primary antibody and secondary antibody (Blue) were used as unlabeled control.

• IP

Unknown

Immunoprecipitation - Anti-Heme Oxygenase 1 antibody [EPR1390Y] (AB68477)

ab68477 (purified) at 1/20 immunoprecipitating Heme Oxygenase 1 in A549 (Human lung carcinoma cell line) whole cell lysate.

Lane 1 (input) : A549 whole cell lysate (10ug).

Lane 2 (+) : ab68477 + A549 whole cell lysate.

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab133267 in HeLa whole cell lysate.

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-Heme Oxygenase 1 antibody [EPR1390Y] (ab68477)

Predicted band size: 33 kDa

Observed band size: 33 kDa

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• WB

Lab

Western blot - Anti-Heme Oxygenase 1 antibody [EPR1390Y] (AB68477)

Lanes 1 - 7 : Merged signal (red and green). Green - ab68477 observed at 33 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab68477 was shown to react with Heme Oxygenase 1 in wild-type A549 cells in Western blot with loss of signal observed in HMOX1 knockout cell line ab269503 (knockout cell lysate ab269665). Wild-type A549 and HMOX1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab68477 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-Heme Oxygenase 1 antibody [EPR1390Y] (ab68477) at 1/10000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

Western blot - Human HMOX1 (Heme Oxygenase 1) knockout A549 cell lysate (<a href='/products/cell-lysates/human-hmox1-heme-oxygenase-1-knockout-a549-cell-lysate-ab269665'>ab269665</a>) at 20 µg

Lane 3:

Human Spleen tissue lysate at 20 µg

Lane 4:

HL-60 cell lysate at 20 µg

Lane 5:

MCF7 cell lysate at 20 µg

Lane 6:

HeLa cell lysate at 20 µg

Lane 7:

A549 cell lysate at 20 µg

Predicted band size: 33 kDa

Observed band size: 33 kDa

false

• WB

Lab

Western blot - Anti-Heme Oxygenase 1 antibody [EPR1390Y] (AB68477)

We are unsure how to define the extra bands.

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

All lanes:

Western blot - Anti-Heme Oxygenase 1 antibody [EPR1390Y] (ab68477) at 1/1000 dilution

Lane 1:

HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates at 20 µg

Lane 2:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates at 20 µg

Lane 3:

A549 (Human lung carcinoma epithelial cell) whole cell lysates at 20 µg

Lane 4:

Mouse spleen lysates 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: 33 kDa

Observed band size: 33 kDa

false

Exposure time: 10s

• WB

Lab

Western blot - Anti-Heme Oxygenase 1 antibody [EPR1390Y] (AB68477)

Hek293 & HL60 presumed negative or very low expression.

Loading control GAPDH at 38kDa

All lanes:

Western blot - Anti-Heme Oxygenase 1 antibody [EPR1390Y] (ab68477) at 1/1000 dilution

Lane 1:

Hek293 at 10 µg

Lane 2:

HL60 at 10 µg

Lane 3:

HeLa at 10 µg

Lane 4:

A549 at 10 µg

Lane 5:

Hu spleen at 10 µg

Lane 6:

Ms spleen at 10 µg

Lane 7:

Rt spleen at 10 µg

Secondary

All lanes:

IRDye® 800CW Goat anti Rabbit

Predicted band size: 33 kDa

Observed band size: 32 kDa

false

• WB

Lab

Western blot - Anti-Heme Oxygenase 1 antibody [EPR1390Y] (AB68477)

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Heme Oxygenase 1 antibody [EPR1390Y] (ab68477) at 1/20000 dilution

Lane 1:

Rat kidney lysate at 20 µg

Lane 2:

Rat spleen lysate at 20 µg

Lane 3:

Mouse kidney 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: 33 kDa

Observed band size: 33 kDa

false