Anti-MLK3 antibody [EP1460Y] - BSA and Azide free

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-MLK3 antibody [EP1460Y] - BSA and Azide free (AB239840)

This data was developed using ab51068 the same antibody clone in a different buffer formulation.
Immunocytochemistry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling MLK3 with Purified ab51068 at 1 : 50 dilution (3.52 μg/ml). Cells were fixed in 100% Methanol and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1 : 200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor® 488,ab150077) was used as the secondary antibody at 1 : 1000 (2 μg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• WB

Lab

Western blot - Anti-MLK3 antibody [EP1460Y] - BSA and Azide free (AB239840)

This data was developed using the same antibody clone in a different buffer formulation (ab51068).

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : MLK3 knockout HAP1 cell lysate (20 μg)
Lane 3 : A431 cell lysate (20 μg)
Lane 4 : HeLa cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab51068 observed at 105 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab51068 was shown to specifically react with MLK3 when MLK3 knockout samples were used. Wild-type and MLK3 knockout samples were subjected to SDS-PAGE. ab51068 and ab8245 (loading control to GAPDH) were diluted 1/5000 and 1/1000 respectively and incubated overnight at 4°C. 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-MLK3 antibody [EP1460Y] - BSA and Azide free (ab239840)

Predicted band size: 93 kDa

false

• WB

Lab

Western blot - Anti-MLK3 antibody [EP1460Y] - BSA and Azide free (AB239840)

This data was developed using the same antibody clone in a different buffer formulation (ab51068).

Lanes 1- 2 : Merged signal (red and green). Green - ab51068 observed at 105 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab51068 was shown to react with MLK3 in wild-type A549 cells in western blot. Loss of signal was observed when knockout cell line ab267169 (knockout cell lysate ab257519) was used. Wild-type A549 and MAP3K11 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. ab51068 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 5000 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-MLK3 antibody [EP1460Y] (<a href='/en-us/products/primary-antibodies/mlk3-antibody-ep1460y-ab51068'>ab51068</a>) at 1/5000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

MAP3K11 knockout A549 cell lysate at 20 µg

Lane 2:

Western blot - Human MAP3K11 (MLK3) knockout A549 cell line (<a href='/en-us/products/cell-lines/human-map3k11-mlk3-knockout-a549-cell-line-ab267169'>ab267169</a>)

Predicted band size: 93 kDa

Observed band size: 105 kDa

false

• WB

Lab

Western blot - Anti-MLK3 antibody [EP1460Y] - BSA and Azide free (AB239840)

This data was developed using the same antibody clone in a different buffer formulation (ab51068).

Lanes 1- 2 : Merged signal (red and green). Green - ab51068 observed at 105 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab51068 was shown to react with MLK3 in wild-type A549 cells in western blot. Loss of signal was observed when knockout cell line ab267168 (knockout cell lysate ab257518) was used. Wild-type A549 and MAP3K11 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. ab51068 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 5000 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-MLK3 antibody [EP1460Y] (<a href='/en-us/products/primary-antibodies/mlk3-antibody-ep1460y-ab51068'>ab51068</a>) at 1/5000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

MAP3K11 knockout A549 cell lysate at 20 µg

Lane 2:

Western blot - Human MAP3K11 (MLK3) knockout A549 cell line (<a href='/en-us/products/cell-lines/human-map3k11-mlk3-knockout-a549-cell-line-ab267168'>ab267168</a>)

Predicted band size: 93 kDa

Observed band size: 105 kDa

false

• WB

Supplier Data

Western blot - Anti-MLK3 antibody [EP1460Y] - BSA and Azide free (AB239840)

This data was developed using ab51068, the same antibody clone in a different buffer formulation.

Blocking buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-MLK3 antibody [EP1460Y] (<a href='/en-us/products/primary-antibodies/mlk3-antibody-ep1460y-ab51068'>ab51068</a>) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 15 µg

Lane 2:

HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate at 15 µg

Lane 3:

A431 (Human epidermoid carcinoma epithelial cell) whole cell lysate at 15 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 1/20000 dilution

Predicted band size: 93 kDa

Observed band size: 100 kDa

false