![Jurkat cells were grown as recommended by the ATCC in RPMI 1640 + 10% fetal bovine serum with glutamine and fungal antibiotic for 3 days. Cells were serum starved overnight (media above without the fetal bovine serum) and either treated with 50 ng/mL PMA and 250 ng/mL calcium ionophore A23187 for ten minutes or left untreated as a control. Cells were fixed and permeabilized as described above. After blocking the cells with 0.1% saponin + 2 % BSA for 20 minutes, the cells were incubated with 20 μL of anti-MAPK [pTpY185/187]FITC for 20 minutes. Cells were analyzed using a FACSCaliber from Becton Dickinson. The purple represents treated cells while the green overlay represents cells serum starved but left untreated.](/media/images/0096226.jpeg)
BP7256F ERK1/2 (p42/44 MAP Kinase) (pThr185/202 + pTyr187/204) antibody
see related secondary antibodiessee all 33 ERK1/2 products
100 Tests / US$ 515
NOVUS BIOLOGICALS
PO Box 802 Littleton, CO 80160, USA
E-Mail: novus@novus-biologicals.com
Homepage: http://www.novus-biologicals.com
PO Box 802 Littleton, CO 80160, USA
E-Mail: novus@novus-biologicals.com
Homepage: http://www.novus-biologicals.com
Quick Overview
Rabbit anti ERK1/2 (p42/44 MAP Kinase) (pThr185/202 + pTyr187/204) -
Synonyms
ERK-1/2 MAPK1/2 P42/P44-MAPK p38 p40 p41 Extracellular signal regulated kinase 1/2, Insulin stimulated MAP2 kinase, Microtubule associated protein 2 kinase, Mitogen activated protein kinase 1/2/3
Product review
Please read our product review about ERK1/2 (p42/44 MAP Kinase) (pThr185/202 + pTyr187/204): Antibodies to MAP-Kinases.
Product Description
Rabbit anti ERK1/2 (p42/44 MAP Kinase) (pThr185/202 + pTyr187/204) -, Presentation: FITC. Product is tested for Westernblot / Immunoblot ( WB ), Flow cytometry ( F ), Immunofluorescence ( IF )
Properties
| Applications | Westernblot / Immunoblot ( WB ), Flow cytometry ( F ), Immunofluorescence ( IF ) |
| Reactivity | Mamalia ( Mam ), Chicken ( Chk ), Drosophila ( Dros ), Fish ( Fish ), Xenopus ( Xen ) |
| Presentation | FITC |
| Host | Rabbit |
| Catalog Number | BP7256F |
| Swiss Prot. No. | P27361 |
| Quantity | 100 Tests |
| Price | US$ 515 |
| Delivery | Worldwide |
| Manufacturer | Acris Antibodies GmbH |
| Datasheet | view  BP7256F.pdf |
Datasheet Extract
| Polyclonal Antibody to ERK1 & 2/MAPK [pTpY185/187] Phosphospecific Antibody - FITC | |
| Immunogen | Chemically synthesized phosphopeptide derived from the region of ERK1 & 2 that contains threonine 202/185 and tyrosine 204/187, based on the human sequence. Swiss Num.: P27361 Remarks: This region is conserved among many species including human, rat, mouse, cow, frog (Xenopus laevis), snail (Aplysia californica), nematode (Caenorhabditis elegans), and fly (Drosophila melanogaster). ERK1 has a Mr of ~44 kDa, while ERK2 has a Mr of ~42 kDa. |
| Format | State: Liquid Ig fraction Purification: Purified from rabbit serum by sequential epitope-specific chromatography. The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated ERK1 & 2 enzymes. The final product is generated by affinity chromatography using an ERK1 & 2-derived peptide that is phosphorylated at threonine 202/185 and tyrosine 204/187, respectively, within the activation loop. BufferSystem: Phosphate buffered saline, pH 7.3, with 1 % bovine serum albumin, containing 0.09 % sodium azide Label: Fluorescein isothiocyanate (FITC). |
| Applications | Immunoflourescence.
Western blot. Flow cytometry (10 μL/test; has been shown to work using Jurkat cells stimulated with 50 ng/mL PMA and 250 ng/mL calcium ionophore A23187 for ten minutes). |
| Specificity | Phosphorylation site-specific antibody selective for the dually phosphorylated form of the extracellular signal-related protein kinase/mitogen-activated protein kinase (ERK1 & 2/MAPK) enzymes containing a phosphate on threonine 202 and tyrosine 204 (human ERK1), and threonine 185 and tyrosine 187 (human ERK2). The antibody has been shown to recognize the endogenous, active forms of ERK1 & 2 in a variety of cell types following treatment by a broad range of extracellular stimuli. Species: Human, rat, mouse, cow, frog (Xenopus laevis), snail (Aplysia californica), nematode (Caenorhabditis elegans), and fly (Drosophila melanogaster). |
| Storage | Store at -80 ºC. Upon initial thawing, aliquot and store at -80 ºC. Avoid repeated freezing and thawing.
Shelf life: one year from despatch. |
| References | Cobb, M.H. (1999) MAP kinase pathways. Prog. Biophys. Mol. Biol. 71(3-4):479-500.
Tan, P.B., and S.K. Kim (1999) Signaling specificity: the RTK/RAS/MAP kinase pathway in metazoans. Trends Genet. 15(4):145-9. Fleisher, T.A. (1999) Rapid Communication: Detection of intracellular phosphorylated STAT-1 by flow cytometry. Clin. Immunol. 90(3):425-430. |
| Protocols | Recommended procedure for staining Jurkat cells with anti-MAPK [pTpY185/187] Phosphospecific Antibody FITC: Buffers:
IC FixTM: 1% paraformaldehyde in 50 mM phosphate buffered saline, pH 7.3. IC PermTM: Dilute the 5x solution of IC PermTM to 50 mM phosphate buffered saline, pH 7.3, 2% (v/v) fetal bovine serum, 0.1% (w/v) sodium azide and 0.1% (w/v) saponin. 100% Methanol Treatment of Jurkat cells: Grow Jurkat cells in RPMI 1640 + 10% fetal bovine serum with glutamine and fungal antibiotic as recommended by the ATCC for 3 days. Serum starve cells overnight (media above without the fetal bovine serum) and either treat with 50 ng/mL PMA and 250 ng/mL calcium ionophore A23187 for ten minutes or leave untreated as a control. Fixation and permeabilization 1. In a 50 mL conical tube wash the cells twice with cold PBS. Temperature control at this point is of vital importance. Cells should be kept on ice any time they are not spinning, and cells should be spun in a temperature controlled centrifuge at 4°C. 2. Fix the cells with 1% paraformaldehyde for 15 minutes at 4°C. It is crucial that the cells are resuspended thoroughly. 3. Wash the cells twice with ice cold PBS. 4. Permeabilize the cells by resuspending them in 100% ice cold methanol added dropwise while the cells are vortexing. Again, it is crucial that the cells are resuspended thoroughly. Leave in methanol for 15 minutes. 5. Wash the cells twice with cold PBS. Antibody incubation 1. Add approximately 1 x 10e6 cells to flow cytometry tubes and wash with 1x IC PermTM. 2. Resuspend cells in 1 mL 1x IC PermTM and allow to sit for 30 minutes blocking at RT. This step is included to reduce the background of the signal. 3. Add 20 μL of antibody in 100 μL of 1x IC PermTM to treated and untreated cells and incubate for 20 minutes at RT. 4. Wash the cells twice, once with 1x IC PermTM and once with PBS. 5. Resuspend the cells in 500 μL of PBS and analyze on a FACS machine such as a FACScan or FACSCaliber. Optional step for a determination of antibody background For a select number of tubes designated as negative controls, incubate 10 μL of anti-MAPK [pTpY185/187] peptide with every 20 μL of anti-MAPK [pTpY185/187] FITC per test prior to incubating the antibody with the cells. In the presence of control peptide, all antibody binding can be attributed to non-specific binding. Gating: Set gates on living cells. Non-viable cells will not show a response. |
| Pictures | Jurkat cells were grown as recommended by the ATCC in RPMI 1640 + 10% fetal bovine serum with glutamine and fungal antibiotic for 3 days. Cells were serum starved overnight (media above without the fetal bovine serum) and either treated with 50 ng/mL PMA and 250 ng/mL calcium ionophore A23187 for ten minutes or left untreated as a control. Cells were fixed and permeabilized as described above. After blocking the cells with 0.1% saponin + 2 % BSA for 20 minutes, the cells were incubated with 20 μL of anti-MAPK [pTpY185/187]FITC for 20 minutes. Cells were analyzed using a FACSCaliber from Becton Dickinson. The purple represents treated cells while the green overlay represents cells serum starved but left untreated. |
12 Secondary Antibodies
| Catalog No. | Name / Host | Presentation | Reactivity | ||||
|---|---|---|---|---|---|---|---|
| R1364B | Rabbit IgG (H&L) | ||||||
| Goat | Biotin | 2 mg / US$ 295 | |||||
| R1364F | Rabbit IgG (H&L) | ||||||
| Goat | FITC | 2 mg / US$ 285 | |||||
| R1364T | Rabbit IgG (H&L) | ||||||
| Goat | TRITC | 2 mg / US$ 285 | |||||
| R1364TR | Rabbit IgG (H&L) | ||||||
| Goat | Texas Red | 2 mg / US$ 295 | |||||
| R1364HRP | Rabbit IgG (H&L) | ||||||
| Goat | HRP | 2 mg / US$ 295 | |||||
| R1364AP | Rabbit IgG (H&L) | ||||||
| Goat | AP | 1 mg / US$ 325 | |||||
| R1458C2 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy2 | 1 mg / US$ 445 | |||||
| R1458C3 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy3 | 1 mg / US$ 445 | |||||
| R1458C35 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy3.5 | 1 mg / US$ 445 | |||||
| R1458C5 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy5 | 1 mg / US$ 445 | |||||
| R1458C55 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy5.5 | 1 mg / US$ 445 | |||||
| R1427R | Rabbit IgG (H&L) F(ab')2 Fragment multi-species ads. | ||||||
| Donkey | PE | 1 ml / US$ 455 | |||||
