- MA1-099 - Invitrogen Antibodies MAPK1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of ERK2 was performed by loading the indicated amounts of ERK1-GST (Product # PV3311) and ERK2-GST (Product # PV3313) human recombinant proteins and 10 µL of PageRuler Plus Prestaned Protein Ladder (Product # 26619) per well onto a Novex 4-20% Tris-glycine polyacrylamide gel (Product # WT4202BOX ). Proteins were transferred to a nitrocellulose membrane using the Power Blotter (Product # 22834), and blocked with 5% milk in TBST for at least 1 hour at room temperature. ERK1 and ERK2 were detected at ~71 kD and 70 kD, respectively, using a p42 MAP Kinase/ERK2/MAPK1 monoclonal antibody (Product # MA1-099) at a dilution of 1:1000 in blocking buffer overnight at 4C on a rocking platform, followed by an HRP-conjugated goat anti-mouse IgG secondary antibody (Product # 31430) at a dilution of 1:40,000 for at least 30 minutes at room temperature. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of ERK2 was performed by loading 30 µg of the indicated lysates and 10 µL of PageRuler Plus Prestaned Protein Ladder (Product # 26619) per well onto a Novex 4-20% Tris-glycine polyacrylamide gel (Product # WT4202BOX ). Proteins were transferred to a nitrocellulose membrane using the Power Blotter (Product # 22834), and blocked with 5% milk in TBST for at least 1 hour at room temperature. ERK2 were detected at ~42 kD using a p42 MAP Kinase/ERK2/MAPK1 monoclonal antibody (Product # MA1-099) at a dilution of 1:1000 in blocking buffer overnight at 4C on a rocking platform, followed by an HRP-conjugated goat anti-mouse IgG secondary antibody (Product # 31430) at a dilution of 1:40,000 for at least 30 minutes at room temperature. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of p42-ERK and phospho-ERK1/2 expression was performed by loading 50 µg HeLa (lane 1 and 2, HeLa (human), NRK (lane 3 and 4, NRK (rat) and MDCK (lane 5 and 6, MDCK (canine) cell lysates with or without TPA treatment onto a 10% Tris-Glycine polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and blocked with 1% BSA/TBST for at least 1 hour at room temperature. p42 ERK was detected using a mouse monoclonal antibody (Product # MA1-099) and phospho-ERK1/2 was detected using a rabbit monoclonal antibody (Product # 700012) at a concentration of 1 µg/mL in blocking buffer overnight at 4°C on a rocking platform. Blots were then incubated with a goat anti-mouse IgG Alexa Fluor790 secondary antibody (Product # A11357, green, top panel) and a goat anti-rabbit IgG Alexa Fluor 680 secondary antibody (Product # A-21109, red, bottom panel) at a dilution of 1:10,000 for at least 1 hour. Fluorescent detection was performed using the Odyssey® CLx imaging system (Li-cor Biosciences). Images generated by Joell Solan in Paul Lampe Lab at Fred Hutchinson Cancer Research Center.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot of MAPK1 was performed by loading 30 µg of WT (lane 1) and MAPK1 CRISPR KO (lane 2) HeLa cell lysates in RIPA buffer onto a 4-15% gradient polyacrylamide gel. Proteins on the blots were visualized with Ponceau staining (below immunoblot). Proteins were transferred to nitrocellulose membrane and blocked in 5% milk for 1 hr. MAPK1 was detected at approximately 41 kDa using a MAPK1 recombinant monoclonal antibody (Product # MA1-099) at a dilution of 1:1000 in 5% BSA in TBS with 0.1% Tween 20 (TBST) overnight at 4 deg. The peroxidase-conjugated secondary antibody (Product # 62-6520) was diluted to 0.2 µg/mL in TBST with 5% milk for 1 hr. Chemiluminescent detection was performed using Pierce ECL Western Blotting Substrate (Product # 32106). Data courtesy of YCharOS Inc., an open science company with the mission of characterizing commercially available antibodies using knockout validation.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of MAPK1 (green) in HeLa cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature. Cells were then blocked with 5% normal goat serum (Product # 31873) for 15 minutes at room temperature. Cells were then probed with a mouse monoclonal antibody recognizing MAPK1 (Product # MA1-099), at a dilution of 1:200 for at least 1 hour at room temperature. Cells were then washed with PBS and incubated with DyLight 488 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 10X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of ERK2 on HeLa cells. Cells were fixed, permeabilized, and stained with a p42 MAP Kinase/ERK2/MAPK1 monoclonal antibody (Product # MA1-099, blue histogram) or a Mouse IgG2a isotype control (Product # MA1-10418, red histogram) at a concentration of 1 µg/mL. After incubation of the primary antibody for at least 1 hour on ice, the cells were stained with a DyLight 488-conjugated goat anti-mouse IgG secondary antibody (Product # 35502) for at least 30 minutes on ice. A representative 10,000 cells were acquired for each sample.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Chromatin immunoprecipitation analysis of ERK2/MAPK1 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 ERK2/MAPK1 monoclonal antibody (Product # MA1-099). 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.

MA1-099