Anti-MCL1 antibody [Y37]

9 product images

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  Western blot - Anti-MCL1 antibody [Y37] (ab32087)

  Lanes 1-3: Merged signal (red and green). Green - ab32087 observed at 37 kDa. Red - loading control Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 observed at 50 kDa.
  

  ab32087 Anti-MCL1 antibody [Y37] was shown to specifically react with MCL1 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line Human MCL1 knockout HEK-293T cell line ab266838 (knockout cell lysate Human MCL1 knockout HEK-293T cell lysate ab256986) was used. Wild-type and MCL1 knockout samples were subjected to SDS-PAGE. ab32087 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-MCL1 antibody [Y37] (ab32087) at 1/1000 dilution

  Lane 1: Wild-type HEK293T cell lysate at 20 µg

  Lane 2: MCL1 knockout HEK293T cell lysate at 20 µg

  Lane 2: Western blot - Human MCL1 knockout HEK-293T cell line (Human MCL1 knockout HEK-293T cell line ab266838)

  Lane 3: Ramos cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/10000 dilution

  Predicted band size: 37 kDa

  Observed band size: 37 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MCL1 antibody [Y37] (ab32087)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon adenocarcinoma tissue labelling MCL1 with purified ab32087 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) ab97051, a goat anti-rabbit IgG H&L (HRP) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

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  Western blot - Anti-MCL1 antibody [Y37] (ab32087)

  MCL1 Western blot staining using rabbit Anti-MCL1 antibody

  Exposure time for samples 1-3: 15 seconds; exposure time for samples 4-6: 1 minute.
  

  Additional bands: We are unsure as to the identity of these extra bands.

  All lanes: Western blot - Anti-MCL1 antibody [Y37] (ab32087) at 1/1000 dilution

  Lane 1: Human lung tissue at 20 µg

  Lane 2: Human lung cancer tissue at 20 µg

  Lane 3: Human liver tissue at 20 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG (H+L), Peroxidase conjugated. at 1/1000 dilution

  Predicted band size: 37 kDa

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  Western blot - Anti-MCL1 antibody [Y37] (ab32087)

  MCL1 Western blot staining using rabbit Anti-MCL1 antibody

  Additional bands: We are unsure as to the identity of these extra bands.

  All lanes: Western blot - Anti-MCL1 antibody [Y37] (ab32087) at 1/1000 dilution

  Lane 1: Ramos (human Burkitt's lymphoma cell line) cell lysate at 20 µg

  Lane 2: HL-60 (human promyelocytic leukemia cell line) cell lysate at 20 µg

  Lane 3: A431 (human epidermoid carcinoma cell line) cell lysate at 20 µg

  Lane 4: HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate at 20 µg

  Lane 5: MCF7 (human breast adenocarcinoma cell line) cell lysate at 20 µg

  Lane 6: HepG2 (human liver hepatocellular carcinoma cell line) cell lysate at 20 µg

  Secondary

  All lanes: Goat anti-rabbit IgG (H+L), peroxidase conjugated. at 1/1000 dilution

  Predicted band size: 37 kDa

  Exposure time: 15s

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  Immunocytochemistry/ Immunofluorescence - Anti-MCL1 antibody [Y37] (ab32087)

  Immunocytochemistry/Immunofluorescence analysis of HCT 116 (human colorectal carcinoma cell line) cells labelling MCL1 with purified ab32087 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, a mouse anti-tubulin (1/1000) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.
  

  Control 1: primary antibody (1/100) and secondary antibody, Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

  Control 2: Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (1/1000) and secondary antibody, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

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  Immunocytochemistry/ Immunofluorescence - Anti-MCL1 antibody [Y37] (ab32087)

  Immunocytochemistry/Immunofluorescence analysis of HCT 116 (human colorectal carcinoma cell line) cells treated with wogonin (Wogonin, COX-2 inhibitor ab142471) labelling MCL1 with unpurified ab32087. Decrease of MCL1 expression correlates with increased concentration of wogonin, as described in literature. Cells were incubated at 37°C for 2h in media containing different concentrations of Wogonin, COX-2 inhibitor ab142471 (wogonin) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab32087 (1/100) dilution was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed ab96899) at 1/250 dilution was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

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  Flow Cytometry (Intracellular) - Anti-MCL1 antibody [Y37] (ab32087)

  Intracellular Flow Cytometry analysis ofRamos (human Burkitt's lymphoma cell line) cells labelling MCL1 with purified ab32087 at 1/250 (red). Cells were fixed with 4% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.
  

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  Flow Cytometry (Intracellular) - Anti-MCL1 antibody [Y37] (ab32087)

  Intracellular Flow Cytometry analysis ofA431 (human epidermoid carcinoma cell line) cells labelling MCL1 with unpurified ab32087 (red line). Cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32087, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed ab96899) at 1/500 dilution for 30 min at 22°C.
  

  Black - Isotype control, rabbit monoclonal IgG.

  Acquisition of >5,000 events was performed. This antibody gave a decreased signal in A431 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween used under the same conditions.

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  Immunocytochemistry/ Immunofluorescence - Anti-MCL1 antibody [Y37] (ab32087)

  Immunocytochemistry/Immunofluorescence analysis of H1299 cells labelling MCL1 with unpurified ab32087. Cells were PFA-fixed and permeabilized in 0.5% Triton X-100 prior to blocking in 3% Serum for 1 hour at 24°C. The primary antibody was diluted 1/100 and incubated with the sample for 1 hour at 24°C. The secondary antibody was an Alexa Fluor^® 488-conjugated Goat anti-Rabbit polyclonal, diluted 1/2000. DAPI (blue) was used as the nuclear counterstain.