- 44-452 - Invitrogen Antibodies MAP2K2 antibody

Choi D, Montermini L, Meehan B, Lazaris A, Metrakos P, Rak J
Journal of extracellular vesicles 2021 Jun;10(8):e12091

Xu J, Keeton AB, Franklin JL, Li X, Venable DY, Frank SJ, Messina JL
The Journal of biological chemistry 2006 Jan 13;281(2):982-92

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Peptide Competition and Stimulation Extracts of NIH3T3 cells untreated (lane 1) or treated with 50 ng/mL PDGF for 15 minutes (2-5) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 4% BSA-TBST buffer overnight at 4°C, then incubated with the MEK1&2 (pS222) antibody for two hours at room temperature in a 1% BSA-TBST buffer, following prior incubation with: no peptide (1, 2), the non-phosphopeptide corresponding to the phosphopeptide immunogen (3), a generic phosphoserine-containing peptide (4), or the phosphopeptide immunogen (5). After washing, the membrane was incubated with goat F (ab’)2 anti-rabbit IgG alkaline phosphatase (Product # ALI4405) and signals were detected using the Pierce SuperSignal™ method. The data show that only the phosphopeptide corresponding to MEK1&2 (pS222) block the antibody signal, demonstrating the specificity of the antibody. The data also show the induction of MEK1&2 (pS222) phosphorylation by the addition of PDGF to this cell system.

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 (Lane 1) and A549 treated with EGF (200 ng/mL for 10 min) (Lane 2) and A549 treated with Afatinib (0.5 uM for 16 h) followed by EGF (200 ng/mL for 10 min). The blots were probed with Anti-Phospho-MEK1/MEK2 (Ser222) Rabbit Polyclonal Antibody (Product # 44-452, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 45 kDa band corresponding to Phospho-MEK1/MEK2 (Ser222) was observed in untreated A549 lysate, the signal increased upon EGF treatment, and decreased upon pretreatment with the EGFR antagonist, Afatinib. 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: MEK1⁄2 (pS222) phosphospecific antibody. Mouse embryonic fibroblasts stained with anti-MEK1⁄2 (pS222) (Product # 44-452). The cell is actively dividing. Blue represents chromosomes in anaphase of mitotic cell division. Green shows mitotic spindle expressing phosphorylated MEK1⁄2.

44-452