Anti-SPHK1 antibody [EPR24511-37]

• WB

Lab

Western blot - Anti-SPHK1 antibody [EPR24511-37] (AB302714)

Western blot : Rabbit Monoclonal[EPR24511-37] to SPHK1 ab302714 staining at 1/1000 dilution, shown in green; Mouse anti alpha Tubulin (ab7291) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 55 kDa in Wild-type A549 cell lysates with no signal observed at this size in SPHK1 knockout A549 cell line (ab301254). To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 100pc LICOR 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 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-SPHK1 antibody [EPR24511-37] (ab302714) at 1/1000 dilution

Lane 1:

Wild-type A549 at 20 µg

Lane 2:

Western blot - Human SPHK1 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-sphk1-knockout-a549-cell-line-ab301254'>ab301254</a>) at 20 µg

Lane 3:

HeLa at 20 µg

Lane 4:

Raji at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 85 kDa

Observed band size: 85 kDa

false

• WB

Supplier Data

Western blot - Anti-SPHK1 antibody [EPR24511-37] (AB302714)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Exposure time : Lane 1 and 2 : 180 seconds Lane 3 : 15 seconds

All lanes:

Western blot - Anti-SPHK1 antibody [EPR24511-37] (ab302714) at 1/1000 dilution

Lane 1:

Human kidney tissue lysate 20 µg

Lane 2:

Human heart tissue lysate 20 µg

Lane 3:

human lu tissue lysate 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/1000 dilution

Observed band size: 42 kDa

false

• WB

Supplier Data

Western blot - Anti-SPHK1 antibody [EPR24511-37] (AB302714)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Exposure time : 70 seconds

All lanes:

Western blot - Anti-SPHK1 antibody [EPR24511-37] (ab302714) at 1/1000 dilution

Lane 1:

U-118 MG (human brain glioma cell), whole cell lysate 20 µg

Lane 2:

SW1353 (human chondrosarcoma fibroblast), whole cell lysate 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 1/50000 dilution

Observed band size: 42 kDa

false

Exposure time: 70s

• WB

Supplier Data

Western blot - Anti-SPHK1 antibody [EPR24511-37] (AB302714)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The additional band at ~50 kDa is expected to be isoform 2 (Q9NYA1-2) of SPHK1 and its expression is also significantly reduced through SPHK1 knockdown. Exposure time : 180 seconds

All lanes:

Western blot - Anti-SPHK1 antibody [EPR24511-37] (ab302714) at 1/1000 dilution

Lane 1:

Hela (human cervix adenocarcinoma epithelial cell) transfected with scrambled siRNA control, whole cell lysate 20 µg

Lane 2:

Hela transfected with siRNA specifically targeti SPHK1, whole cell lysate 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 1/50000 dilution

Observed band size: 42 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-SPHK1 antibody [EPR24511-37] (AB302714)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Exposure time : 70 seconds

All lanes:

Western blot - Anti-SPHK1 antibody [EPR24511-37] (ab302714) at 1/1000 dilution

Lane 1:

Hela (human cervix adenocarcinoma epithelial cell), whole cell lysate 20 µg

Lane 2:

HepG2 (human hepatocellar carcinoma epithelial cell), whole cell lysate 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 1/50000 dilution

Observed band size: 42 kDa

false

Exposure time: 70s