44470G

antibody from Invitrogen Antibodies

Targeting: MAP2K6 MAPKK6, MEK6, MKK6, PRKMK6, SAPKK3

Western blot (Supportive data in Antibodypedia)

Antibody Data

Product number

44470G

Provider

Invitrogen Antibodies

Product name

Phospho-MEK3/MEK6 (Ser189, Thr193, Ser207, Thr211) Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Reactivity

Human

Host

Rabbit

Isotype

IgG

Vial size

100 µL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on whole cell extracts (20 µg lysate) of A549 (lane 1), A549 treated for 20 minutes with 200 nM of PMA (lane 2), A549 treated for 30 minutes with 25 µg/mL of Anisomycin (lane 3) and A549 treated for 40 minutes with UV (lane 4). The blots were probed with Anti-MEK3 (pS189/pT193) + MEK6 (pS207/pT211) Rabbit Polyclonal Antibody (Product # 44470G, 1:500-1:2000 dilution) and detected by chemiluminescence Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). Two bands ~ 37 and 39 kDa corresponding to MEK3 (pS189/pT193) + MEK6 (pS207/pT211) was observed in Anisomycin treated cell line tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), 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

Mal-E-tagged fusion protein expressing MEK6 left inactivated (6) or activated with MEKK (1-5) was spiked into background extracts and resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer overnight at 4oC, then incubated with the MEK3/6 (pSpT189/193)/ (pSpT207/211) antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 6), the non-phosphopeptide corresponding to the phosphopeptide immunogen (2), a generic hosphoserine-containing peptide (3), a generic phosphothreonine-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 Tropix WesternStar™ method. The data show that only the phosphopeptide corresponding to MEK3/6 (pSpT189/193)/ (pSpT207/211) blocks the antibody signal, demonstrating the specificity of the antibody. Please note that the Mal-E-tagged fusion protein expressing MEK6 runs at ~81 kDa.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Mal-E-tagged fusion protein expressing MEK6 left inactivated (6) or activated with MEKK (1-5) was spiked into background extracts and resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer overnight at 4oC, then incubated with the MEK3/6 (pSpT189/193)/ (pSpT207/211) antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 6), the non-phosphopeptide corresponding to the phosphopeptide immunogen (2), a generic hosphoserine-containing peptide (3), a generic phosphothreonine-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 Tropix WesternStar™ method. The data show that only the phosphopeptide corresponding to MEK3/6 (pSpT189/193)/ (pSpT207/211) blocks the antibody signal, demonstrating the specificity of the antibody. Please note that the Mal-E-tagged fusion protein expressing MEK6 runs at ~81 kDa.