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

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : MCL1 knockout HEK293T cell lysate
Lane 3 : Ramos cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 37 kDa
Observed band size: 37 kDa

Lanes 1-3: Merged signal (red and green). Green - ab32087 observed at 37 kDa. Red - 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 ab266838 (knockout 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 (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 (ab216773) and 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.

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. 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.

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. 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. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

Flow Cytometry analysis of Ramos (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.

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

Lane 1 : Human lung tissue with NFDM/TBST
Lane 2 : Human lung cancer tissue with NFDM/TBST
Lane 3 : Human liver tissue with NFDM/TBST

Lysates/proteins at 20 µg per lane.

Blocking peptides at 5 % per lane.

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

Predicted band size: 37 kDa

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

Lane 1 : Ramos (human Burkitt's lymphoma cell line) cell lysate with NFDM/TBST
Lane 2 : HL-60 (human promyelocytic leukemia cell line) cell lysate with NFDM/TBST
Lane 3 : A431 (human epidermoid carcinoma cell line) cell lysate with NFDM/TBST
Lane 4 : HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate with NFDM/TBST
Lane 5 : MCF7 (human breast adenocarcinoma cell line) cell lysate with NFDM/TBST
Lane 6 : HepG2 (human liver hepatocellular carcinoma cell line) cell lysate with NFDM/TBST

Lysates/proteins at 20 µg per lane.

Blocking peptides at 5 % per lane.

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

Predicted band size: 37 kDa


Exposure time: 15 seconds

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

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.

See Abreview

Flow Cytometry analysis of A431 (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) (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.

Immunocytochemistry/Immunofluorescence analysis of HCT 116 (human colorectal carcinoma cell line) cells treated with wogonin (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 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 (ab96899) at 1/250 dilution was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.