Recombinant Anti-mSin3a antibody [EPR27103-53] (ab307197)

All lanes : Anti-mSin3a antibody [EPR27103-53] (ab307197) at 1/1000 dilution

Lane 1 : Wild-type HAP1(human chronic myelogenous leukemia near-haploid cell line) whole cell lysate
Lane 2 : mSin3A knockout HAP1 whole cell lysate
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (ab216773) at 1/10000 dilution

Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.

Lysates at 20 µg per lane.

The samples were run on a Bis-Tris gel.

 

Performed under reducing conditions.

False colour image of Western blot: Anti-mSin3A antibody (ab307197) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody 6C5 (ab8245) loading control staining at 1/20000 dilution, shown in red.

In Western blot, ab307197 was shown to bind specifically to mSin3A. A band was observed at 170 kDa in wild-type HAP1 cell lysates with no signal observed at this size in mSin3A knockout cell line. To generate this image, wild-type and mSin3A knockout HAP1 cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in in Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS 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 IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes : Anti-mSin3a antibody [EPR27103-53] (ab307197) at 1/1000 dilution

Lane 1 : Human testis tissue lysate
Lane 2 : Mouse testis tissue lysate
Lane 3 : K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate
Lane 4 : 293T (human embryonic kidney epithelial cell) whole cell lysate
Lane 5 : F9 (mouse embryonal carcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

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

The identity of the bands below 75 kDa are unknown.

The blot was developed using a higher sensitivity ECL substrate.

Exposure time: 180 seconds.

All lanes : Anti-mSin3a antibody [EPR27103-53] (ab307197) at 1/1000 dilution

Lane 1 : C6 (rat glial tumor glial cell) whole cell lysate 20 µg
Lane 2 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

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

The identity of the bands below 75 kDa are unknown.

This blot was developed using a high sensitivity ECL substrate.

Exposure time: 180 seconds.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) (Right panel) compared to mSin3A knockout HAP1 (Left panel) cells labeling mSin3A with AB307197 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue).

A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized F9 (mouse embryonal carcinoma epithelial cell) cells labeling mSin3A with AB307197 at 1/500 dilution (0.1ug) (Red) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

mSin3A was immunoprecipitated from 0.35 mg HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate 10 µg with AB307197 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using AB307197 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HAP1 whole cell lysate 10 µg

Lane 2: AB307197 IP in HAP1 whole cell lysate 10 ug

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab307197 in HAP1 cell lysate

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

Exposure time: 6 seconds.