711257

antibody from Invitrogen Antibodies

Targeting: MAP2K3 MAPKK3, MEK3, MKK3, PRKMK3

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

711257

Provider

Invitrogen Antibodies

Product name

MEK3 Recombinant Polyclonal Antibody (1HCLC)

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with Monkey, Dog,Cat and Mouse.

Antibody clone number

1HCLC

Concentration

0.5 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on Whole cell extracts (30 µg lysate) of U-2 OS (Lane 1), Caco-2 (Lane 2), U-87 MG (Lane 3), Hep G2 (Lane 4), PC-3 (Lane 5), HeLa (Lane 6) and A-431 (Lane 7). The blots were probed with Anti-MKK3 Recombinant Rabbit Polyclonal Antibody (Product # 711257, 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). Two isoforms of 39 kDa and 36 kDa band corresponding to MKK3 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).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-MEK3 Polyclonal Antibody (Product # 711257) and bands at 39 kDa and 36 kDa corresponding to MEK3 was observed across cell lines tested. Whole cell extracts (30 µg lysate) of Hep G2 (Lane 1), U-87 MG (Lane 2), IMR-32 (Lane 3) and HeLa (Lane 4) 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 (1µg/ml) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036), 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

For immunofluorescence analysis, C2C12 cells were fixed and permeabilized for detection of endogenous MKK3 using MKK3 Recombinant Rabbit Polyclonal Antibody (Product # 711257, 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 MKK3 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 cytoplasmic localization of MKK3. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.