Anti-MEKK2 抗体 [EP626Y]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MEKK2 antibody [EP626Y] (AB33918)

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.

• WB

Lab

Western blot - Anti-MEKK2 antibody [EP626Y] (AB33918)

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.

All lanes:

Western blot - Anti-MEKK2 antibody [EP626Y] (ab33918) at 1/10000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

MAP3K2 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human MAP3K2 (MEKK2) knockout HeLa cell line (<a href='/products/cell-lines/human-map3k2-mekk2-knockout-hela-cell-line-ab264944'>ab264944</a>)

Predicted band size: 70 kDa

Observed band size: 75 kDa

false

• WB

Lab

Western blot - Anti-MEKK2 antibody [EP626Y] (AB33918)

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.

All lanes:

Western blot - Anti-MEKK2 antibody [EP626Y] (ab33918) at 1/10000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

MAP3K2 knockout A549 cell lysate at 20 µg

Lane 2:

Western blot - Human MAP3K2 (MEKK2) knockout A549 cell line (<a href='/products/cell-lines/human-map3k2-mekk2-knockout-a549-cell-line-ab267153'>ab267153</a>)

Predicted band size: 70 kDa

Observed band size: 75 kDa

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• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-MEKK2 antibody [EP626Y] (AB33918)

ab33918 staining MEKK2 in Jurkat (human acute T cell leukemia) cellsby intracellular 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)

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-MEKK2 antibody [EP626Y] (AB33918)

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 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. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MEKK2 antibody [EP626Y] (AB33918)

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-MEKK2 antibody [EP626Y] (AB33918)

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)

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MEKK2 antibody [EP626Y] (AB33918)

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.

• IP

Supplier Data

Immunoprecipitation - Anti-MEKK2 antibody [EP626Y] (AB33918)

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.

All lanes:

Immunoprecipitation - Anti-MEKK2 antibody [EP626Y] (ab33918)

Predicted band size: 70 kDa

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• IP

Supplier Data

Immunoprecipitation - Anti-MEKK2 antibody [EP626Y] (AB33918)

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

All lanes:

Immunoprecipitation - Anti-MEKK2 antibody [EP626Y] (ab33918)

Predicted band size: 70 kDa

false

• WB

Supplier Data

Western blot - Anti-MEKK2 antibody [EP626Y] (AB33918)

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:

Western blot - Anti-MEKK2 antibody [EP626Y] (ab33918)

Predicted band size: 70 kDa

false

• WB

Lab

Western blot - Anti-MEKK2 antibody [EP626Y] (AB33918)

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:

Western blot - Anti-MEKK2 antibody [EP626Y] (ab33918) at 1/10000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

MAP3K2 knockout A549 cell lysate at 20 µg

Lane 2:

Western blot - Human MAP3K2 (MEKK2) knockout A549 cell line (<a href='/products/cell-lines/human-map3k2-mekk2-knockout-a549-cell-line-ab267152'>ab267152</a>)

Predicted band size: 70 kDa

Observed band size: 75 kDa

false

• WB

Lab

Western blot - Anti-MEKK2 antibody [EP626Y] (AB33918)

All lanes:

Western blot - Anti-MEKK2 antibody [EP626Y] (ab33918) at 1/20000 dilution

All lanes:

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

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 70 kDa

false

• WB

Lab

Western blot - Anti-MEKK2 antibody [EP626Y] (AB33918)

All lanes:

Western blot - Anti-MEKK2 antibody [EP626Y] (ab33918) at 1/20000 dilution

Lane 1:

C6 (rat glioma) whole cell lysate at 20 µg

Lane 2:

NIH/3T3 (mouse embryo) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 70 kDa

false

• WB

Lab

Western blot - Anti-MEKK2 antibody [EP626Y] (AB33918)

All lanes:

Western blot - Anti-MEKK2 antibody [EP626Y] (ab33918) at 1/20000 dilution

Lane 1:

Jurkat (human acute T cell leukemia) whole cell lysate at 20 µg

Lane 2:

HeLa (human cervix adenocarcinoma) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 70 kDa

Observed band size: 70 kDa

false