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Western blot
ImmunocytochemistryAntibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [1]
- Immunocytochemistry [1]
- Immunohistochemistry [1]
- Chromatin Immunoprecipitation [1]
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- Product number
- GTX15780 - Provider product page
- Provider
- GeneTex
- Product name
- ERK1 antibody [12D11]
- Antibody type
- Monoclonal
- Reactivity
- Human, Mouse, Simian
- Host
- Mouse
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Supportive validation
- Submitted by
- GeneTex (provider)
- Main image
- Experimental details
- Western blot analysis of ERK1 in 25ug of various cell lysates. Proteins were transferred to a PVDF membrane and blocked with 5% Milk/TBST for at least 1 hour. Membranes were probed with p44 MAP Kinase/ERK1/MAPK3 antibody [12D11] at a dilution of 1:1000 overnight at 4¢XC on a rocking platform. Membranes were washed in TBS-0.1%Tween 20 and probed with a HRP-conjugated secondary antibody. Membranes were washed and chemiluminescent detection was performed.
Enhanced validation
- Submitted by
- GeneTex (provider)
- Enhanced method
- Genetic validation
- 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 for 15 minutes at room temperature. Cells were probed with p44 MAP Kinase/ERK1/MAPK3 antibody [12D11] at a dilution of 1:100 for at least 1 hour at room temperature. Cells were washed with PBS and incubated with DyLight 549-conjugated secondary antibody. Nuclei (blue) were stained with Hoechst 33342 dye. Images were taken at 20X magnification.
Supportive validation
- Submitted by
- GeneTex (provider)
- Main image
- 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¢XC. Following antigen retrieval tissues were blocked in 10% normal goat serum/TBS for two hours at room temperature. Tissues were probed at a dilution of 1:100 with p44 MAP Kinase/ERK1/MAPK3 antibody [12D11] overnight at 4¢XC in a humidified chamber. Tissues were washed extensively with TBS/0.025% Triton and endogenous peroxidase activity quenched. Detection was performed using a HRP-conjugated secondary antibody followed by colorimetric detection using metal enhanced DAB. Images were taken at 10X magnification.
Supportive validation
- Submitted by
- GeneTex (provider)
- Main image
- Experimental details
- Chromatin immunoprecipitation analysis of ERK1/MAPK3 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 with 1.0ul/100ul well volume of p44 MAP Kinase/ERK1/MAPK3 antibody [12D11]. Chromatin aliquots from ~1 x 105 cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1ul of eluted DNA in 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.
