720309

antibody from Invitrogen Antibodies

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

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Flow cytometry (Recommended by provider)

Antibody Data

Product number

720309

Provider

Invitrogen Antibodies

Product name

MEK6 Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Reactivity

Human, Mouse

Host

Rabbit

Isotype

IgG

Vial size

100 µg

Concentration

0.5 mg/mL

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

Knockdown of MEK6 was achieved by transfecting HeLa with MEK6 specific siRNAs (Silencer® select Product # s11180 and s11181). Western blot analysis (Fig. a) was performed using Whole Cell Extract-WCL from the MEK6 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with MEK6 Polyclonal Antibody (Product # 720309, 0.75 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to MEK6.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-MEK6 Polyclonal Antibody(Product # 720309) and a 38kDa band corresponding to MEK6 was observed across cell lines and tissues tested except mouse heart. Whole Cell Extract-WCL (30 µg lysate) of HeLa (Lane 1), A549 (Lane 2), SH-SY5Y (Lane 3), MCF7 (Lane 4), A-431 (Lane 5), Mouse Skeletal Muscle (Lane 6) and Mouse Heart (Lane 7) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (0.75 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on Whole cell extracts (30 µg lysate) of HeLa (Lane 1), NIH/3T3 (Lane 2), MOLT4 (Lane 3), A549 (Lane 4), SH-SY 5Y (Lane 5), NTERA-2 (Lane 6), Hep G2 (Lane 7), PBMC (Lane 8) and HEK-293 (Lane 9). The blots were probed with Anti-MKK6 Rabbit Polyclonal Antibody (Product # 720309, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 38 kDa band corresponding to MKK6 was observed across the cell lines tested. 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).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

For immunofluorescence analysis, SHSY-5Y cells were fixed and permeabilized for detection of endogenous MKK6 using Anti- MKK6 Rabbit Polyclonal Antibody (Product # 720309, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of MKK6 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating nuclear localization of MKK6. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.