Anti-Acid sphingomyelinase antibody [EPR23090-181]

• WB

Lab

Western blot - Anti-Acid sphingomyelinase antibody [EPR23090-181] (AB272729)

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

The molecular weight observed is consistent with what has been described in the literature (PMID : 25803076, 25853898).

ASM expression can be induced by PMA in THP-1 cells (PMID : 10224156).

Exposure time : 3 minutes.

All lanes:

Western blot - Anti-Acid sphingomyelinase antibody [EPR23090-181] (ab272729) at 1/1000 dilution

Lane 1:

Untreated THP-1 (human monocytic leukemia monocyte) whole cell lysate at 40 µg

Lane 2:

THP-1 treated with 80nM phorbol-12-myristate-13-acetate (PMA) (<a href='/en-us/products/biochemicals/phorbol-12-myristate-13-acetate-pma-pkc-activator-ab120297'>ab120297</a>) overnight whole cell lysate at 40 µg

Lane 3:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 40 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 70 kDa

Observed band size: 75 kDa

false

• WB

Lab

Western blot - Anti-Acid sphingomyelinase antibody [EPR23090-181] (AB272729)

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

This blot was developed using a higher sensitivity ECL substrate.

Exposure time : 3 minutes.

All lanes:

Western blot - Anti-Acid sphingomyelinase antibody [EPR23090-181] (ab272729) at 1/500 dilution

All lanes:

Human heart tissue lysate at 10 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 70 kDa

Observed band size: 75 kDa

false

• WB

Lab

Western blot - Anti-Acid sphingomyelinase antibody [EPR23090-181] (AB272729)

ab272729 was shown to react with SMPD1 in wild-type U-87 MG cell culture media in Western blot with loss of signal observed in a SMPD1 siRNA knockdown cell culture media. Cell culture media from wild-type U-87 MG transfected with either scrambled siRNA or SMPD1 siRNA were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab272729 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-Acid sphingomyelinase antibody [EPR23090-181] (ab272729) at 1/1000 dilution

Lane 1:

Wild-type U-87 MG transfected with scrambled siRNA control cell culture media at 10 µg

Lane 2:

U-87 MG transfected with siRNA specifically targeting SMPD1 cell culture media at 10 µg

Observed band size: 75 kDa

false

• WB

Lab

Western blot - Anti-Acid sphingomyelinase antibody [EPR23090-181] (AB272729)

Western blot : Rabbit Monoclonal[EPR23090-181] to Acid sphingomyelinase ab272729 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 74 kDa in Wild-type MCF7 cell lysates with no signal observed at this size in SMPD1 knockout MCF7 cell line. To generate this image, 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 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Acid sphingomyelinase antibody [EPR23090-181] (ab272729) at 1/1000 dilution

Lane 1:

Wild-type MCF7 at 20 µg

Lane 2:

SMPD1 knockout MCF7 at 20 µg

Lane 3:

HeLa at 20 µg

Lane 4:

Ramos at 20 µg

Secondary

All lanes:

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

Predicted band size: 70 kDa

Observed band size: 74 kDa

false