Anti-MBD4/MED1 antibody [EPR28263-16]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-MBD4/MED1 antibody [EPR28263-16] (AB315462)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized MBD4 KO HeLa (human MBD4 knockout cervical adenocarcinoma epithelial cell, Left) / Parental HeLa (Right) cells labelling MBD4/MED1 with ab315462 at 1/50 dilution (1 ug)/Red compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• WB

Supplier Data

Western blot - Anti-MBD4/MED1 antibody [EPR28263-16] (AB315462)

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

Lysates were freshly made and used immediately to minimize protein degradation.

The band below 75 kDa may be caused by degradation.

The identity of the lower MW band at approximately 25 kDa is unknown.

All lanes:

Western blot - Anti-MBD4/MED1 antibody [EPR28263-16] (ab315462) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

THP-1 (human monocytic leukemia monocyte) whole cell lysate at 20 µg

Lane 3:

Jurkat (human T cell leukemia T lymphocyte) whole cell lysate at 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 1/100000 dilution

Observed band size: 75 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-MBD4/MED1 antibody [EPR28263-16] (AB315462)

Western blot : Anti-MBD4 antibody (ab315462) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody 6C5 loading control (ab8245) staining at 1/20000 dilution, shown in red.

In Western blot, ab315462 was shown to bind specifically to MBD4. Target of interest was observed at 75 kDa in wild-type HeLa cell lysates (lane 1) with no signal observed at this size in MBD4 knockout cell line (lane 2, knockout cell line ab264937/ knockout cell lysate ab258960). To generate this image, samples were first run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in a fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed in TBS-T, incubated with secondary antibodies Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution for 1 h at room temperature, washed again then imaged.

Lysate was freshly made and used for Western blotting immediately to minimize protein degradation.

All lanes:

Western blot - Anti-MBD4/MED1 antibody [EPR28263-16] (ab315462) at 1/1000 dilution

Lane 1:

Wild type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg with Intercept® (TBS) Blocking Buffer diluted with an equal volume of TBS

Lane 2:

MBD4 knockout HeLa whole cell lysate at 20 µg with Intercept® (TBS) Blocking Buffer diluted with an equal volume of TBS

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

Observed band size: 75 kDa,36 kDa

false

Exposure time: 180s