All lanes : Anti-MEKK2 antibody [EP626Y] (ab33918) at 1/10000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : MAP3K2 knockout A549 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 70 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab33918 observed at 75 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab33918 was shown to react with MEKK2 in wild-type A549 cells in western blot. Loss of signal was observed when knockout cell line ab267153 (knockout cell lysate ab257522) was used. Wild-type A549 and MAP3K2 knockout A549 cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab33918 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 10000 dilution and a 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.

ab33918 staining MEKK2 in human colon carcinoma tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1: PBS in place of primary antibody.

 

ab33918 staining MEKK2 in MCF-7 (human breast carcinoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000. ab7291 and ab150120 were used as counterstains for primary antibody ab75748 and secondary antibody ab150077 respectively and DAPI was used as a nuclear counterstain.

Negative control 1: Rabbit primary antibody and anti-mouse secondary antibody (ab150120)
Negative control 2: Mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab150077)

 

ab33918 staining MEKK2 in Jurkat (human acute T cell leukemia) cells by flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/120. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/500 was used as the secondary antibody.

Isoytype control: Rabbit monoclonal IgG (Black)

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

 

All lanes : Anti-MEKK2 antibody [EP626Y] (ab33918) at 1/10000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : MAP3K2 knockout A549 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 70 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab33918 observed at 75 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab33918 was shown to react with MEKK2 in wild-type A549 cells in western blot. Loss of signal was observed when knockout cell line ab267152 (knockout cell lysate ab257521) was used. Wild-type A549 and MAP3K2 knockout A549 cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab33918 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 10000 dilution and a 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.

All lanes : Anti-MEKK2 antibody [EP626Y] (ab33918) at 1/10000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : MAP3K2 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 70 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab33918 observed at 75 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab33918 was shown to react with MEKK2 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264944 (knockout cell lysate ab257520) was used. Wild-type HeLa and MAP3K2 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab33918 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 10000 dilution and a 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.

ab33918 immunoprecipitating MEKK2. 10µg of cell lysate was incubated with primary antibody at a dilution of 1/40 and VeriBlot for IP Detection Reagent (HRP) (ab131366) at a dilution of 1/10000.

Lane 1: HepG2 (human hepatocellular carcinoma) whole cell lysate (10ug)
Lane 2: HepG2 (human hepatocellular carcinoma) whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab33918 in HepG2 (human hepatocellular carcinoma) whole cell lysate

 

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: MEKK2 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: Human breast carcinoma lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab33918 observed at 75 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab33918 was shown to recognize MEKK2 when MEKK2 knockout samples were used, along with additional cross-reactive bands. Wild-type and MEKK2 knockout samples were subjected to SDS-PAGE. ab33918 and ab8245 (loading control to GAPDH) were both diluted 1/10 000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) andGoat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

All lanes : Anti-MEKK2 antibody [EP626Y] (ab33918) at 1/20000 dilution

Lane 1 : C6 (rat glioma) whole cell lysate
Lane 2 : NIH/3T3 (mouse embryo) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 dilution

Predicted band size: 70 kDa

K562 (human chronic myelogenous leukemia) whole cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 dilution

Predicted band size: 70 kDa

All lanes : Anti-MEKK2 antibody [EP626Y] (ab33918) at 1/20000 dilution

Lane 1 : Jurkat (human acute T cell leukemia) whole cell lysate
Lane 2 : HeLa (human cervix adenocarcinoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 dilution

Predicted band size: 70 kDa
Additional bands at: 70 kDa. We are unsure as to the identity of these extra bands.

ab33918 staining MEKK2 in mouse cerebral cortex tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1: PBS in place of primary antibody.

ab33918 staining MEKK2 in rat cerebral cortex tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1: PBS in place of primary antibody.

Overlay histogram showing HepG2 cells stained with unpurified ab33918 (red line). The cells were fixed with 80% 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 (ab33918, 1/50 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

MEKK2 was immunoprecipitated using 0.5mg HepG2 whole cell extract, 10µg of unpurified Rabbit monoclonal [EP626Y] to MEKK2 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, HepG2 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab33918.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697) .
Band: 75kDa: MEKK2.