Anti-Acid sphingomyelinase antibody [EPR23090-181] - BSA and Azide free

• WB

Lab

Western blot - Anti-Acid sphingomyelinase antibody [EPR23090-181] - BSA and Azide free (AB273510)

This data was developed using the same antibody clone in a different buffer formulation (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 (ab326026). 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] (<a href='/en-us/products/primary-antibodies/acid-sphingomyelinase-antibody-epr23090-181-ab272729'>ab272729</a>) at 1/1000 dilution

Lane 1:

Wild-type MCF7 at 20 µg

Lane 2:

Western blot - Human SMPD1 knockout MCF7 cell line (<a href='/en-us/products/cell-lines/human-smpd1-knockout-mcf7-cell-line-ab326026'>ab326026</a>) 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

• WB

Lab

Western blot - Anti-Acid sphingomyelinase antibody [EPR23090-181] - BSA and Azide free (AB273510)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, glycerol, BSA and sodium azide (ab272729).

All lanes:

Western blot - Anti-Acid sphingomyelinase antibody [EPR23090-181] (<a href='/en-us/products/primary-antibodies/acid-sphingomyelinase-antibody-epr23090-181-ab272729'>ab272729</a>) 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] - BSA and Azide free (AB273510)

This data was developed using the same antibody clone in a different buffer formulation (ab272729)

ab273510 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] (<a href='/en-us/products/primary-antibodies/acid-sphingomyelinase-antibody-epr23090-181-ab272729'>ab272729</a>) 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