- PA1-4631 - Invitrogen Antibodies MAP2K1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot of MEK1 in human T47D cells showing specific immunolabeling of a band at ~45 kDa corresponding to Phospho-MEK1 (Thr386) polyclonal antibody (Product # PA1-4631) in the first lane (-). Phosphospecificity is shown in the second lane (+) where immunolabeling is completely eliminated by blot treatment with lambda phosphatase (1,200 units for 30 min).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of A549 (1), A549 treated with EGF (200 ng/mL for 10 minutes) (2), A-431 (3), A-431 treated with EGF (200 ng/mL for 10 minutes) (4) and A-431 treated with Afatinib followed by EGF (0.5 uM of Afatinib for 6 hours, 200 ng/mL of EGF for 10 minutes) (5). The blot was probed with Anti-Phospho-MEK1 (Thr386) Rabbit Polyclonal Antibody (Product # PA1-4631, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 45 kDa band corresponding to Phospho-MEK1 (Thr386) was detected. Pre-treatment with EGFR-antagonist, Afatinib, resulted in inhibition of Phospho-MEK1 (Thr386) in A-431 cell line. 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® 2 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: Immunofluorescence analysis of MEK1 (Thr386) was performed using 90% confluent log phase A-431 cells treated with 200 ng/mL of EGF for 10 minutes. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Phospho-MEK1 (Thr386) Rabbit Polyclonal Antibody (Product # PA1-4631) at 1:250 dilution in 0.1% BSA and incubated overnight at 4 degree Celsius and then labelled with Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:100). Panel d represents the merged image showing cytoplasmic localization. Panel e represents cells treated with antagonist, Afatinib (1uM for 6hrs) followed by EGF (200 ng/mL for 10 minutes), showing no signal. Panel f shows untreated cells with weak cytoplasmic localization. Panel g represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

PA1-4631