710318

antibody from Invitrogen Antibodies

Targeting: MAPK3 ERK1, p44erk1, p44mapk, PRKM3

Western blot (Supportive data in Antibodypedia)

Chromatin Immunoprecipitation (Supportive data in Antibodypedia)

Antibody Data

Product number

710318

Provider

Invitrogen Antibodies

Product name

ERK1/ERK2 Recombinant Polyclonal Antibody (6HCLC)

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with mouse, rat, non-human primate and rabbit based on sequence homology.

Antibody clone number

6HCLC

Concentration

0.5 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of p44/42 MAPK (ERK1/2) (pT202/pY204) was performed by loading 20 µg of NIH/3T3 (lane1) and NIH/3T3 treated for 10 minutes with 100 nM/mL of EGF (lane2) cell lysates using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800), and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for overnight at 4°C. p44/42 MAPK (ERK1/2) (pT202/pY204) was detected at ~42 kDa using p44/42 MAPK (ERK1/2) (pT202/pY204) Recombinant Rabbit Polyclonal Antibody (Product # 710318) at 0.5-1 µg/mL in 2.5% skim milk at room temperature for 3 hours on a rocking platform. Goat anti-Rabbit IgG - HRP Secondary Antibody (Product # G-21234) at 1:5000 dilution was used and 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 ERK1/ERK2 in whole cell extracts from NIH-3T3 cells treated with PDGF using an ERK1/ERK2 Recombinant Rabbit Polyclonal Antibody (Product # 710318) at a dilution of 1 µg/mL. To confirm specificity, competition was performed by preincubation with the phosphopeptide to inhibit antibody binding (lane 2). Samples were detected using chemiluminescence (ECL). Results show a band at ~42kDa.

Chromatin Immunoprecipitation

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Chromatin immunoprecipitation analysis of ERK1+ERK23 was performed using cross-linked chromatin from 1 x 10^6 HCT116 human colon carcinoma cells treated with serum for 0, 15, 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+ERK23 Recombinant Rabbit Polyclonal Antibody (Product # 710318). Chromatin aliquots from ~1 x 10^5 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 -3.2kb upstream of the human FOS gene, or exon-4 of human FOS. 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 FOS locus is shown above the data where boxes represent exons (grey boxes = translated regions, white boxes = untranslated regions), the zigzag lines represent introns, and the straight line represents upstream sequence. Regions amplified by FOS primers are represented by black bars. Data courtesy of the Innovators Program.