- MA1-13041 - Invitrogen Antibodies MAPK3 antibody

Submitted references Engineering microdeletions and microduplications by targeting segmental duplications with CRISPR.

Tai DJ, Ragavendran A, Manavalan P, Stortchevoi A, Seabra CM, Erdin S, Collins RL, Blumenthal I, Chen X, Shen Y, Sahin M, Zhang C, Lee C, Gusella JF, Talkowski ME
Nature neuroscience 2016 Mar;19(3):517-22

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of ERK1 was performed by loading 25 µg of various whole cell lysates onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% Milk/TBST for at least 1 hour. Membranes were probed with a mouse monoclonal antibody recognizing ERK1 (Product # MA1-13041) at a dilution of 1:1000 overnight at 4°C on a rocking platform. Membranes were washed in TBS-0.1%Tween 20 and probed with a goat anti-mouse-HRP secondary antibody (Product # 32430) at a dilution of 1:25,000 for at least one hour. Membranes were washed and chemiluminescent detection performed using Pierce Super Signal West Pico (Product # 34077).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of ERK1 (green) in untreated and siRNA knockdown treated HeLa cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 15 minutes at room temperature. Cells were then blocked with 5% normal goat serum (Product # 31873) for 15 minutes at room temperature. Cells were probed with a mouse monoclonal antibody recognizing ERK1 (Product # MA1-13041), at a dilution of 1:100 for at least 1 hour at room temperature. Cells were washed with PBS and incubated with DyLight 549 goat-anti-mouse secondary antibody at a dilution of 1:400 for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of ERK1 was performed using 70% confluent log phase A549 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 ERK1 (12D11) Mouse Monoclonal Antibody (Product # MA1-13041) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) 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 shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on cancer biopsies of deparaffinized human colon tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer for 20 minutes at 95°C. Following antigen retrieval tissues were blocked in 10% normal goat serum (Product # 31873)/TBS (Product # 28376) for two hours at room temperature. Tissues were probed at a dilution of 1:100 with a mouse monoclonal antibody recognizing ERK1 (Product # MA1-13041) overnight at 4°C in a humidified chamber. Tissues were washed extensively with TBS/0.025% Triton (Product # 28314) and endogenous peroxidase activity quenched with Peroxidase Suppressor (Product # 35000) for 30 minutes at room temperature. Detection was performed using a goat anti-mouse HRP secondary antibody (Product # 31430) at a dilution of 1:500 followed by colorimetric detection using metal enhanced DAB (Product # 34065). Images were taken on a Zeiss Axiovision microscope at 10X magnification. Results demonstrate strong cytoplasmic localization of ERK1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Chromatin immunoprecipitation analysis of ERK1 was performed using cross-linked chromatin from 1 x 106 HCT116 colon carcinoma cells treated with serum for 0, 15, 30, and 60 minutes. Immunoprecipitation was performed using a multiplex microplate Matrix ChIP assay (see reference for Matrix ChIP protocol: http://www.ncbi.nlm.nih.gov/pubmed/22098709) with 1.0 µL/100 µL well volume of an ERK1 monoclonal antibody (Product # MA1-13041). Chromatin aliquots from ~1 x 105 cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1 µL of eluted DNA in 2 µL SYBR real-time PCR reactions containing primers to amplify -15kb upstream of the Egr1 gene or exon-1 of Egr1. PCR calibration curves were generated for each primer pair from a dilution series of sheared total genomic DNA. Quantitation of immunoprecipitated chromatin is presented as signal relative to the total amount of input chromatin. Results represent the mean +/- SEM for three experiments. A schematic representation of the Egr-1 locus is shown above the data where boxes represent exons (black boxes = translated regions, white boxes = untranslated regions); the zigzag line represents an intron; and the straight line represents upstream sequence. Regions amplified by Egr-1 primers are represented by black bars. Data courtesy of the Innovators Program.

MA1-13041

Antibody data