Anti-mSin3a antibody [EPR27103-53]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-mSin3a antibody [EPR27103-53] (AB307197)

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.

• IP

Supplier Data

Immunoprecipitation - Anti-mSin3a antibody [EPR27103-53] (AB307197)

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.

All lanes:

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

All lanes:

HAP1 whole cell lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 170 kDa

false

Exposure time: 6s

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-mSin3a antibody [EPR27103-53] (AB307197)

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.

• WB

Supplier Data

Western blot - Anti-mSin3a antibody [EPR27103-53] (AB307197)

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:

Western blot - Anti-mSin3a antibody [EPR27103-53] (ab307197) at 1/1000 dilution

Lane 1:

Human testis tissue lysate at 20 µg

Lane 2:

Mouse testis tissue lysate at 20 µg

Lane 3:

K-562 whole cell lysate at 20 µg

Lane 4:

293T whole cell lysate at 20 µg

Lane 5:

F9 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: 170 kDa

true

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-mSin3a antibody [EPR27103-53] (AB307197)

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:

Western blot - Anti-mSin3a antibody [EPR27103-53] (ab307197) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

mSin3A knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Secondary

Lanes 1 - 3:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Lanes 1 - 3:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Observed band size: 170 kDa

false

• WB

Lab

Western blot - Anti-mSin3a antibody [EPR27103-53] (AB307197)

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

All lanes:

Western blot - Anti-mSin3a antibody [EPR27103-53] (ab307197) at 1/1000 dilution

Lane 1:

K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 2:

Human testis tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 170 kDa

false

Exposure time: 40s

• WB

Supplier Data

Western blot - Anti-mSin3a antibody [EPR27103-53] (AB307197)

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. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-mSin3a antibody [EPR27103-53] (ab307197) at 1/1000 dilution

Lane 1:

C6 whole cell lysate at 20 µg

Lane 2:

RAW264.7 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: 170 kDa

true

Exposure time: 180s