Anti-Alas1 antibody [EPR10247] - Mitochondrial Marker

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Alas1 antibody [EPR10247] - Mitochondrial Marker (AB154860)

Immunocytochemistry/Immunofluorescence analysis of HepG2 cells labeling Alas1 with unpurified ab154860 at a dilution of 1/250.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Alas1 antibody [EPR10247] - Mitochondrial Marker (AB154860)

Immunocytochemistry/Immunofluorescence analysis of HepG2 cells labelling Alas1 with purified ab154860 at a dilution of 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1 : primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2 : ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

• WB

Lab

Western blot - Anti-Alas1 antibody [EPR10247] - Mitochondrial Marker (AB154860)

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Alas1 antibody [EPR10247] - Mitochondrial Marker (ab154860) at 1/10000 dilution

All lanes:

HepG2 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 1/100000 dilution

Predicted band size: 31 kDa,45 kDa,71 kDa,82 kDa

Observed band size: 31 kDa,40 kDa,48 kDa,71 kDa

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• WB

Lab

Western blot - Anti-Alas1 antibody [EPR10247] - Mitochondrial Marker (AB154860)

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Alas1 antibody [EPR10247] - Mitochondrial Marker (ab154860) at 1/10000 dilution

All lanes:

Raji 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 1/100000 dilution

Predicted band size: 71 kDa

Observed band size: 71 kDa

false

• WB

Unknown

Western blot - Anti-Alas1 antibody [EPR10247] - Mitochondrial Marker (AB154860)

Lanes 1-4 : Merged signal (red and green). Green - ab154860 observed at 71 kDa. Red - loading control ab8245 observed at 36 kDa.

ab154860 Anti-Alas1 antibody [EPR10247] was shown to specifically react with Alas1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab266473 (knockout cell lysate ab257348) was used. Wild-type and Alas1 knockout samples were subjected to SDS-PAGE. ab154860 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C 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-Alas1 antibody [EPR10247] - Mitochondrial Marker (ab154860) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

ALAS1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human ALAS1 knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-alas1-knockout-hek-293t-cell-line-ab266473'>ab266473</a>)

Lane 3:

HepG2 cell lysate at 20 µg

Lane 4:

HEK-293 cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 71 kDa

Observed band size: 71 kDa

false

• WB

Unknown

Western blot - Anti-Alas1 antibody [EPR10247] - Mitochondrial Marker (AB154860)

All lanes:

Western blot - Anti-Alas1 antibody [EPR10247] - Mitochondrial Marker (ab154860) at 1/1000 dilution

Lane 1:

HepG2 cell lysate at 10 µg

Lane 2:

K562 cell lysate at 10 µg

Lane 3:

JAR cell lysate at 10 µg

Lane 4:

Raji cell lysate at 10 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 71 kDa

false