- 702581 - Invitrogen Antibodies MAP2K1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on Whole cell extracts (30 µg lysate) of HEK-293 (Lane 1), HEK-293 treated with EGF (100 ng/mL for 30 min) (Lane 2), NIH/3T3 (Lane 3), NIH/3T3 treated with EGF (100 ng/mL for 30 min) (Lane 4), A-431 (Lane 5), A-431 treated with EGF (100 ng/mL for 30 min) (Lane 6), U-87 MG (Lane 7) and U-87 MG treated with EGF (100 ng/mL for 30 min) (Lane 8). The blots were probed with Anti-Phospho-MEK1 (Thr386) Recombinant Rabbit Monoclonal Antibody (Product # 702581, 0.5-1 µg/mL). A 45 kDa band corresponding to MEK (pT386) was increased upon EGF treatment in all the cell lines tested. The blots were detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:5000 dilution). Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: For immunofluorescence analysis, A431 cells were fixed and permeabilized for detection of endogenous MEK1 pT386 using Anti- Phospho-MEK1 (Thr386) Recombinant Rabbit Monoclonal Antibody (Product # 702581, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Nuclei (blue) were stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938), and Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Detection and localization of Phospho-MEK1 (Thr386) (green) in the nucleus can be clearly observed in interphase cells (a-d). When cell cycle progresses to early prophase, MEK1 pT386 can be visualized detaching from the chromatin and associating with the nuclear membrane (e-h). Further progression to late prophase and metaphase results in reorganization of the nuclear membrane, wherein complete detachment of MEK1 pT386 from chromatin and translocation to the cytoplasm can be observed (i-p).The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: For immunofluorescence analysis, A431 cells were fixed and permeabilized for detection of endogenous MEK1 pT386 using Anti- Phospho-MEK1 (Thr386) Recombinant Rabbit Monoclonal Antibody (Product # 702581, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Nuclei (blue) were stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938), and Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Detection and localization of MEK pT386 (green) in the cytoplasm can be clearly observed in metaphase cells (a-d). Antibody specificity was demonstrated by competition with the Phospho-MEK1 (Thr386) phospho peptide, which results in loss of signal detection. No competition was observed with the non-phospho peptide. The images were captured at 60X magnification.

702581