Anti-SOD2/MnSOD antibody [EPR2560Y]

• WB

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Western blot - Anti-SOD2/MnSOD antibody [EPR2560Y] (AB68155)

Lanes 1 - 4 : Merged signal (red and green). Green - ab68155 observed at 29 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab68155 was shown to specifically react with SOD2 when SOD2 knockout samples were used. Wild-type and SOD2 knockout samples were subjected to SDS-PAGE. ab68155 and ab8245 (loading control to GAPDH) were diluted at 1/1000 and 1/10 000 respectively and incubated overnight at 4°C. 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 1/10000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-SOD2/MnSOD antibody [EPR2560Y] (ab68155) at 1/1000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

SOD2 knockout HAP1 cell lysate at 20 µg

Lane 3:

Human heart tissue lysate at 20 µg

Lane 4:

Rat heart tissue lysate at 20 µg

Predicted band size: 24 kDa

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• WB

Lab

Western blot - Anti-SOD2/MnSOD antibody [EPR2560Y] (AB68155)

Lanes 1 - 4 : Merged signal (red and green). Green - ab68155 observed at 22 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab68155 was shown to react with SOD2/MnSOD in wild-type HEK-293T. Loss of signal was observed when knockout cell line ab255434 (knockout cell lysate ab263835) was used. Wild-type and SOD2/MnSOD knockout samples were subjected to SDS-PAGE. ab68155 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-SOD2/MnSOD antibody [EPR2560Y] (ab68155) at 1/1000 dilution

Lane 1:

SHSY5Y cell lysate at 20 µg

Lane 2:

A549 cell lysate at 20 µg

Lane 2:

Western blot - Human SOD2 (MnSOD) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-sod2-mnsod-knockout-hek-293t-cell-line-ab255434'>ab255434</a>)

Lane 3:

Wild-type HEK-293T cell lysate at 20 µg

Lane 4:

SOD2 knockout HEK-293T 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 1/20000 dilution

Predicted band size: 24 kDa

Observed band size: 29 kDa,37 kDa

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• WB

Unknown

Western blot - Anti-SOD2/MnSOD antibody [EPR2560Y] (AB68155)

All lanes:

Western blot - Anti-SOD2/MnSOD antibody [EPR2560Y] (ab68155) at 1/500 dilution

Lane 1:

Rat brain lysate at 10 µg

Lane 2:

SH-SY5Y lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 24 kDa

Observed band size: 25 kDa

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• WB

Lab

Western blot - Anti-SOD2/MnSOD antibody [EPR2560Y] (AB68155)

This blot was produced using 4-20% SDS-PAGE containing 15 μg of rat brain lysate per lane at 150V for 1hr before being transferred onto a 0.45 μm PVDF membrane at 75V for 1hr. The membrane was then blocked for 1hr using 5% NFDM/TBST, then incubated with ab68155 (1/1000) at room temperature for 1hr. After being washed three times in TBST, the membrane was incubated with Peroxidase conjugated goat anti-rabbit IgG (H+L) (ab97051) at 1/100,000 dilution for 1hr at room temperature. The membrane was washed three times again. Then the signal was developed using the ECL technique. ab68155 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in WB. The image shows the band intensity remains relatively constant across all storage temperatures, demonstrating that antibody activity is not affected.

All lanes:

Western blot - Anti-SOD2/MnSOD antibody [EPR2560Y] (ab68155) at 1/1000 dilution

All lanes:

Rat brain lysate at 15 µg with NDFM/TBST

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

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Exposure time: 7s