- PA5-16556 - Invitrogen Antibodies MAP2K1 antibody

Submitted references C/D box snoRNA SNORD113-6/AF357425 plays a dual role in integrin signalling and arterial fibroblast function via pre-mRNA processing and 2'O-ribose methylation.

van Ingen E, van den Homberg DAL, van der Bent ML, Mei H, Papac-Milicevic N, Kremer V, Boon RA, Quax PHA, Wojta J, Nossent AY
Human molecular genetics 2022 Mar 31;31(7):1051-1066

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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 HT-29 (Lane 1), A-431 (Lane 2), A549 (Lane 3), MDA-MB-231 (Lane 4), U-87 MG (Lane 5) and Raji (Lane 6). The blots were probed with Anti-MEK1 Rabbit Polyclonal Antibody (Product # PA5-16556, 1 µg/mL) 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 MEK1 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® 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)
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Experimental details: Western blot analysis of MEK1 / MAP2K1 was performed using whole cell extracts (30 µg lysate) of HeLa (Lane 1), Jurkat (Lane 2), A431 (Lane 3), K562 (Lane 4), SH-SY5Y (Lane 5), PC-12 (Lane 6), A549 (Lane 7), and MCF7 (Lane 8). The blots were probed with Anti-MEK1 / MAP2K1 Rabbit Polyclonal Antibody (Product # PA5-16556, 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 45 kDa band corresponding to p53 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX), 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)
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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 Polyclonal Antibody (Product # PA5-16556, 1 µg/mL dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036) 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)
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Experimental details: Immunofluorescence analysis of MEK1 was performed using 70% confluent log phase A431 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with MEK1 Rabbit Polyclonal Antibody (Product # PA5-16556) at 5 µg/mL in 0.1% BSA and incubated overnight at 4 degree Celsius and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 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 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of MEK1/MAP2K1 was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with MEK1/MAP2K1 Rabbit Polyclonal Antibody (Product # PA5-16556) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) a dilution of 1:2000 for 45 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® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing punctated cytoplasmic and nuclear localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of MEK1/MAP2K1 showing staining in the cytoplasm and nucleus of paraffin-embedded human breast carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a MEK1/MAP2K1 Rabbit Polyclonal Antibody (Product # PA5-16556) diluted in 3% BSA-PBS at a dilution of 1:20 for 1 hour at 37°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of MEK1/MAP2K1 showing staining in the cytoplasm and nucleus of paraffin-embedded human prostate carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a MEK1/MAP2K1 Rabbit Polyclonal Antibody (Product # PA5-16556) diluted in 3% BSA-PBS at a dilution of 1:20 for 1 hour at 37°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of MEK1/MAP2K1 showing staining in the cytoplasm and nucleus of paraffin-embedded mouse lung tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a MEK1/MAP2K1 Rabbit Polyclonal Antibody (Product # PA5-16556) diluted in 3% BSA-PBS at a dilution of 1:20 for 1 hour at 37°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of MEK1 / MAP2K1 was performed using Jurkat cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with MEK1 / MAP2K1 Rabbit Polyclonal Antibody (PA5-16556, red histogram) or with rabbit isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control..

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Protein expression in murine and human primary fibroblasts. ( A ) Western blots for JAG1, DUSP7, ITGB3 and MAP2K1, each with GAPDH, of three independent replicates (each band represents a pool of three technical triplicates) in PMFs. ( B ) Western blots for JAG1, DUSP7, ITGB3 and MAP2K1, with GAPDH of three independent replicates (each band represents a pool of three technical triplicates) in HUAFs. ( C - L ) Quantifications of protein band intensities, normalized to GAPDH. Data are represented as mean +- SEM. * * P < 0.01, compared with GM-ctrl.

PA5-16556

Antibody data