Invitrogen

Phospho-MEK1 (Ser217, Ser221) Recombinant Rabbit Monoclonal Antibody (13H22L22)

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Phospho-MEK1 (Ser217, Ser221) Antibody in Western Blot (WB) — Western blot analysis of MAP2K1 (pSpS217/221) was performed by loading 20 µg of A431 (lane1) and Jurkat (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 1 hour at room temperature. MAP2K1 (pSpS217/221) was detected at ~44 kDa using MAP2K1 (pSpS217/221) Recombinant Rabbit Monoclonal Antibody (Product # 701267) at 1-3 µg/mL in 2.5 % skim milk at 4°C overnight on a rocking platform. To confirm specificity, the corresponding blot on right was incubated with Lambda phosphatase and its reactivity with antibody was tested. 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).

Phospho-MEK1 (Ser217, Ser221) Antibody in ChIP assay (ChIP) — Chromatin immunoprecipitation analysis of Phospho-MEK1/MAP2K1 (pSer217+221) was performed using cross-linked chromatin from 1 x 10^6 HCT116 human colon carcinoma cells treated with serum for 0, 15, and 30 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 a Phospho-MEK1/MAP2K1 rabbit monoclonal antibody (Product # 701267). 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 -15kb upstream of the human Egr-1 locus, or exon-1 or exon-2 of Egr-1. 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.

Product Details

701267

Applications	Tested Dilution	Publications
Western Blot (WB)	1-3 µg/mL	-
ChIP assay (ChIP)	1 µL	-

Product Specifications
Species Reactivity	Human
Host/Isotype	Rabbit / IgG
Class	Recombinant Monoclonal
Type	Antibody
Clone	13H22L22
Immunogen	Phosphopeptide corresponding to amino acids 212–226 of human MAP2K1 [pSpS217/221] View antigen
Conjugate	Unconjugated
Form	Liquid
Concentration	0.5 mg/mL
Purification	Protein A
Storage buffer	PBS
Contains	0.09% sodium azide
Storage conditions	Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.
RRID	AB_2532449

Product Specific Information

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

Intact IgG appears on a non-reducing gel as ~150 kDa band and upon reduction generating a ~25 kDa light chain band and a ~50 kDa heavy chain.

Recombinant rabbit monoclonal antibodies are produced using in vitro expression systems. The expression systems are developed by cloning in the specific antibody DNA sequences from immunoreactive rabbits. Then, individual clones are screened to select the best candidates for production. The advantages of using recombinant rabbit monoclonal antibodies include: better specificity and sensitivity, lot-to-lot consistency, animal origin-free formulations, and broader immunoreactivity to diverse targets due to larger rabbit immune repertoire.

Target Information

MEK1/mitogen activated protein kinase/ERK kinase 1/microtubule-associated protein 2 kinase is a dual specificity kinase and catalyzes both a threonine and a tyrosine residue on MAP kinases ERK1 and ERK2. Mek1 is activated by phosphorylation of serine 218 and 222 residues by Raf1. It is known to be involved in the signaling during stress activate response, apoptosis and proliferative induction by cytokines. Mutations in the gene can lead to cardiofaciocutaneous syndrome.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

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Bioinformatics

Protein Aliases: Dual specificity mitogen-activated protein kinase kinase 1; EC 2.7.12.2; ERK activator kinase 1; kinase MEK1; MAP kinase kinase 1; MAPK/ERK kinase 1; MAPKK 1; MEK 1; MKK (Thr292); MKK (Thr386); MKK2; MP2K1; PRKMK2; protein kinase, mitogen-activated, kinase 1 (MAP kinase kinase 1)

Gene Aliases: CFC3; MAP2K1; MAPKK1; MEK1; MKK1; PRKMK1

UniProt ID: (Human) Q02750

Entrez Gene ID: (Human) 5604

Molecular Function: non-receptor serine/threonine protein kinase