- 13-3500 - Invitrogen Antibodies MAP2K1 antibody

Brobeil A, Kämmerer F, Tag C, Steger K, Gattenlöhner S, Wimmer M
Biomolecules 2015 Apr 16;5(2):485-504

Wang HY, Kanungo J, Malbon CC
The Journal of biological chemistry 2002 Feb 1;277(5):3530-6

Greulich H, Erikson RL
The Journal of biological chemistry 1998 May 22;273(21):13280-8

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of MEK1 was performed by loading 30 µg of cell lysate of mouse thymocytes untreated (lane 1) and treated with 2 µM AZD6244 for 18 hr at 37C (lane 2) in 1xSDS sample buffer and SeeBlue Plus2 Pre-stained Protein Standard (Product # LC5925) onto a NuPAGE Novex 4-12% Bis-Tris Protein Gels (Product #NP0324BOX). Proteins were transferred to nitrocellulose membrane of Trans-Blot Turbo Mini Nitrocellulose Transfer Packs using the Trans-Blot Turbo at 15-min 1.3A setting. Membrane was blocked in 1xDPBS (Product #14190136) containing 0.05% Tween 20 and 50% SeaBlock buffer (Product # 37527) for one hour at room temperature. MEK1 was detected at 43kDa molecular weights using a MEK1 antibody (Product # 13-3500) at a dilution of 1:1,000 in 1xDPBS (Product # 14190136) containing 0.05% Tween 20 and 50% SeaBlock buffer for one hour at room temperature on a rocking platform, followed by a Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody conjugated with Alexa Fluor Plus 680 (Product# A32729) at a dilution of 1:10,000 for one hour at room temperature. Fluorescent detection was performed using LI-COR Odyssey imager.

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), H-1975 (Lane 2), HCT 116 (Lane 3), HT-29 (Lane 4), A-431 (Lane 5), A549 (Lane 6), MDA-MB-231 (Lane 7), U-87 MG (Lane 8), MCF7 (Lane 9), Raji (Lane 10) and NIH/3T3 (Lane 11). The blot was probed with Anti-MEK1 Mouse Monoclonal Antibody (Product # 13-3500, 1 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). A 45 kDa band corresponding to MEK1 was detected across all 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® 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: Western blot analysis of MEK1 was performed by loading 20 µg of the indicated whole cell lysates per well and 5 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) onto a 4-20% Tris-Glycine polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane using the G2 Blotter (Product # 62288), and blocked with 5% milk in TBST for 1 hour at room temperature. MEK1 was detected at ~43 kD using a MEK1 monoclonal antibody (Product # 13-3500) at a dilution of 1 µg/mL in 5% milk in TBST overnight at 4C on a rocking platform. After washing, blots were probed with a goat anti-mouse IgG-HRP secondary antibody (Product # 31430) at a dilution of 1:10,000 for at least 30 minutes at room temperature. Chemiluminescent detection was performed using SuperSignal West Pico substrate (Product # 34080).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockout of MEK1 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR934388_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of MEK1 was performed by loading 30 µg of HeLa Cas9 (Lane 1), andHeLa MEK1 KO (Lane 2) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 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 Anti-MEK1 Monoclonal Antibody (3D9) (Product # 13-3500, 1:1,000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:5,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to MEK1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of MEK1 was performed by loading 30 µg of cell lysate of mouse thymocytes untreated (lane 1) and treated with 2 µM AZD6244 for 18 hr at 37C (lane 2) in 1xSDS sample buffer and SeeBlue® Plus2 Pre-stained Protein Standard (Product # LC5925) onto a NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gels (Product #NP0324BOX). Proteins were transferred to nitrocellulose membrane and blocked in 1xDPBS (Product #14190136) containing 0.05% Tween 20 and 50% SeaBlock buffer (Product # 37527) for one hour at room temperature. MEK1 was detected at 43kDa molecular weights using a MEK1 antibody (Product # 13-3500) at a dilution of 1:1,000 in 1xDPBS (Product # 14190136) containing 0.05% Tween 20 and 50% SeaBlock buffer for one hour at room temperature on a rocking platform, followed by a Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody conjugated with Alexa Fluor Plus 680 (Product # A32729) at a dilution of 1:10,000 for one hour at room temperature. Data courtesy of Ab Data Exchange Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of MEK1 was done on 70% confluent log phase MCF7 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes; permeabilized with 0.25% Triton™ X-100 for 10 minutes followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with MEK1 Mouse Monoclonal Antibody (Product # 13-3500) at 2 µg - 4 µg in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor® 488 Rabbit Anti-Mouse IgG Secondary Antibody (Product # A-11059) at a dilution of 1:400 for 30 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 Alexa Fluor® 594 Phalloidin (Product # A12381). Panel d is a merged image showing cytoplasmic localization of MEK1. Panel e shows no primary antibody. The images were captured at 20X magnification.

13-3500