The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
1/5000. Detects a band of approximately 100 kDa (predicted molecular weight: 93 kDa).
1/100 - 1/250.
Application notesIs unsuitable for IHC-Fr,IHC-P or IP.
FunctionActivates the JUN N-terminal pathway. Required for serum-stimulated cell proliferation and for mitogen and cytokine activation of MAPK14 (p38), MAPK3 (ERK) and MAPK8 (JNK1). Plays a role in mitogen-stimulated phosphorylation and activation of BRAF, but does not phosphorylate BRAF directly. Influences microtubule organization during the cell cycle.
Tissue specificityExpressed in a wide variety of normal and neoplastic tissues including fetal lung, liver, heart and kidney, and adult lung, liver, heart, kidney, placenta, skeletal muscle, pancreas and brain.
Sequence similaritiesBelongs to the protein kinase superfamily. STE Ser/Thr protein kinase family. MAP kinase kinase kinase subfamily. Contains 1 protein kinase domain. Contains 1 SH3 domain.
Post-translational modificationsAutophosphorylation on serine and threonine residues within the activation loop plays a role in enzyme activation. Thr-277 is likely to be the main autophosphorylation site. Phosphorylation of Ser-555 and Ser-556 is induced by CDC42.
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.
Western blot - Anti-MLK3 antibody [EP1460Y] (ab51068)
Anti-MLK3 antibody [EP1460Y] (ab51068) at 1/5000 dilution + A431 cell lysate at 10 µg
Secondary Goat anti-Rabbit HRP labeled at 1/2000 dilution