![<b>Peptide Competition</b>
Lysates from PC12 cells unstimulated (1) or stimulated with 0.5 M sorbitol for 5 minutes (2-6) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer overnight at 4oC, and then incubated with the ERK1&2 [pTpY185/187] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following its prior incubation with: no peptide (1, 2), the non-phosphopeptide corresponding to the phosphopeptide immunogen (3), a generic phosphothreonine-containing peptide (4), a generic phosphotyrosine-containing peptide (5), or the phosphopeptide immunogen (6). After washing, the membrane was incubated with goat F(ab)2 anti-rabbit IgG HRP conjugate and signals were detected using the Pierce SuperSignal method.
The data show that only the phosphopeptide corresponding to ERK1&2 [pTpY185/187] blocks the antibody signal, demonstrating the specificity of the antibody. The data also show the induction of ERK1&2 [pTpY185/187] phosphorylation by the addition of sorbitol in this cell system.](/media/images/0092803.jpeg)
BP7256 ERK1/2 (p42/44 MAP Kinase) pThr185/202 + pTyr187/204 antibody
see related secondary antibodies
0.1 ml / please contact our distributor
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: Aff - Purified. Product is tested for Frozen sections ( C ), Immunofluorescence ( IF ), Westernblot / Immunoblot ( WB )
Properties
| Applications | Frozen sections ( C ), Immunofluorescence ( IF ), Westernblot / Immunoblot ( WB ) |
| Reactivity | Mamalia ( Mam ), Chicken ( Chk ), Drosophila ( Dros ), Fish ( Fish ), Xenopus ( Xen ) |
| Presentation | Aff - Purified |
| Host | Rabbit |
| Catalog Number | BP7256 |
| Swiss Prot. No. | P27361 |
| Quantity | 0.1 ml |
| Price | please contact our distributor |
| Delivery | Worldwide |
| Manufacturer | Acris Antibodies GmbH |
| Datasheet | view  BP7256.pdf |
Datasheet Extract
| Polyclonal Antibody to ERK1 & 2/MAPK [pTpY185/187] Phosphospecific Antibody | |
| Background | ERK (extracellular signal-regulated kinase), also known as MAPK (mitogen-activated protein kinase) has two closely related isoforms of 44 kDa and 42 kDa, respectively. These kinases belong to a family of serine/threonine kinases that are activated upon treatment of cells with a large variety of stimuli including mitogens, hormones, growth factors, cytokines, and bioactive peptides. Cell stimulation induces the activation of a signaling cascade, the downstream effects of which have been linked to the regulation of cell growth, differentiation and the cytoskeleton. ERK1 and ERK2 are fully activated by phosphorylation within a Thr-Glu-Tyr motif of the activation loop on both a threonine and a tyrosine residue by MEKs (MAPK/ERK kinases), thereby greatly elevating the activity of the ERK1&2 enzymes. |
| Immunogen | The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of human ERK1/2 that contains threonine 202/185 and tyrosine 204/187. Swiss Num.: P27361 |
| Format | State: Liquid Ig fraction Purification: Sequential epitope-specific chromatography. The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the sites of phosphorylation to remove antibody that is reactive with non-phosphorylated ERK1&2. 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: Dulbecco's phosphate buffered saline (without Mg2+ and Ca2+), pH 7.3 (+/- 0.1), 50% glycerol with 1.0 mg/mL BSA (IgG, protease free) as a carrier and 0.05% sodium azide as preservative |
| Applications | The antibody has been used in Western blotting. For Western blotting applications, we recommend using the antibody at a 1:1000 starting dilution. Positive control used: PC12 cells +/- Sorbitol; NIH3T3 +/- PDGF; HEK293 +/- UV.
Previous lots of this antibody have been used in immunostaining. |
| Specificity | This antibody reacts with ERK1 & 2. Species: Human. |
| Add. Information | The immunizing region is conserved among many species including rat, mouse, cow, frog, snail, nematode, and fruit fly. |
| Storage | Store at -20°C. We recommend a brief centrifugation before opening to settle vial contents. Then, apportion into working aliquots and store at -20°C.
Shelf life: one year from despatch. For shipment or short-term storage (up to one week), 2-8°C is sufficient. |
| References | Karasewski, L. and A. Ferreira (2003) MAPK signal transduction pathway mediates agrin effects on neurite elongation in cultured hippocampal neurons. J. Neurobiol. 55(1):14-24 (cites the use of BP7256 and BP7136).
Janne, P.A., et al. (2002) Inhibition of epidermal growth factor receptor signaling in malignant pleural mesothelioma. Cancer Res. 62(18):5242-5247 (cites the use of this antibody). Robinson, F.L., et al. (2002) Identification of novel point mutations in ERK2 that selectively disrupt binding to MEK1. J. Biol. Chem. 277(17):14844-14852 (cites the use of this antibody). Grammatikakis, N., et al. (2002) The role of Hsp90N, a new member of the Hsp90 family, in signal transduction and neoplastic transformation. J. Biol. Chem. 277(10):8312-8320 (cites the use of this antibody). Janulis, M., et al. (2001) A novel mitogen-activated protein kinase is responsive to Raf and mediates growth factor specificity. Mol. Cell. Biol. 21(6):2235-2247 (cites the use of BP7060, BP7059, BP7256, BP7136, BP7087 and BP7113). Nanki, T., et al. (2001) Chemokines regulate IL-6 and IL-8 production by fibroblast-like synoviocytes from patients with rheumatoid arthritis. J. Immunol. 167(9):5381-5385 (cites the use of BP7215, BP7256, and BP7136). Tong, T., et al. (2001) Involvement of the MAP kinase pathways in induction of GADD45 following UV radiation. Exp. Cell Res. 269(1):64-72 (cites the use of BP7256 and BP7136). Cobb, M.H. (1999) MAP kinase pathways. Prog. Biophys. Mol. Biol. 71(3-4):479-500. Fanger, G.R. (1999) Regulation of the MAPK family members: role of subcellular localization and architectural organization. Histol. Histopathol. 14(3):887-894. Xu, S., et al. (1997) Differential regulation of mitogen-activated protein/ERK kinase (MEK)1 and MEK2 and activation by a Ras-independent mechanism. Mol. Endocrinol. 11(11):1618-1625. |
| Protocols | Western Blotting Procedure
1.Lyse approximately 10e7 cells in 0.5 mL of ice cold Cell Lysis Buffer (formulation provided below). This buffer, a modified RIPA buffer, is suitable for recovery of most proteins, including membrane receptors, cytoskeletal-associated proteins, and soluble proteins. This cell lysis buffer formulation is available as a separate product which requires supplementation with protease inhibitors immediately prior to use. Other cell lysis buffer formulations, such as Laemmli sample buffer and Triton-X 100 buffer, are also compatible with this procedure. Additional optimization of the cell stimulation protocol and cell lysis procedure may be required for each specific application. 2. Remove the cellular debris by centrifuging the lysates at 14,000 x g for 10 minutes. Alternatively, lysates may be ultracentrifuged at 100,000 x g for 30 minutes for greater clarification. 3. Carefully decant the clarified cell lysates into clean tubes and determine the protein concentration using a suitable method, such as the Bradford assay. Polypropylene tubes are recommended for storing cell lysates. 4. React an aliquot of the lysate with an equal volume of 2x Laemmli Sample Buffer (125 mM Tris, pH 6.8, 10% glycerol, 10% SDS, 0.006% bromophenol blue, and 130 mM dithiothreitol [DTT]) and boil the mixture for 90 seconds at 100°C. 5.Load 10-30 µg of the cell lysate into the wells of an appropriate single percentage or gradient minigel and resolve the proteins by SDS-PAGE. 6. In preparation for the Western transfer, cut a piece of PVDF membrane slightly larger than the gel. Soak the membrane in methanol for 1 minute, then rinse with ddH2O for 5 minutes. Alternatively, nitrocellulose may be used. 7. Soak the membrane, 2 pieces of Whatman paper, and Western apparatus sponges in transfer buffer (formulation provided below) for 2 minutes. 8. Assemble the gel and membrane into the sandwich apparatus. 9. Transfer the proteins at 140 mA for 60-90 minutes at room temperature. 10. Following the transfer, rinse the membrane with Tris buffered saline for 2 minutes. 11. Block the membrane with blocking buffer (formulation provided below) for one hour at room temperature or overnight at 4°C. 12. Incubate the blocked blot with primary antibody at a 1:1000 dilution in Tris buffered saline supplemented with 3% Ig-free BSA and 0.1% Tween 20 overnight at 4°C or for two hours at room temperature. 13. Wash the blot with several changes of Tris buffered saline supplemented with 0.1% Tween 20. 14. Detect the antibody band using an appropriate secondary antibody, such as goat F(ab')2 anti-rabbit IgG alkaline phosphatase conjugate or goat F(ab')2 anti-rabbit IgG horseradish peroxidase conjugate in conjunction with your chemiluminescence reagents and instrumentation. BUFFER FORMULATIONS Cell lysis buffer: 10 mM Tris, pH 7.4 100 mM NaCl 1 mM EDTA 1 mM EGTA 1 mM NaF 20 mM Na4P2O7 2 mM NaVO4 0.1% SDS 0.5% sodium deoxycholate 1% Triton-X 100 10% glycerol 1 mM PMSF (made from a 0.3 M stock in DMSO)or 1 mM AEBSF (water soluble version of PMSF) 60 µg/mL aprotinin 10 µg/mL leupeptin 1 µg/mL pepstatin (alternatively, protease inhibitor cocktail such as Sigma Cat. # P2714 may be used) Transfer buffer: 2.4 gm Tris base 14.2 gm glycine 200 mL methanol Q.S. to 1 liter, then add 1 mL 10% SDS. Cool to 4°C prior to use. Tris buffered saline: 20 mM Tris-HCl, pH 7.4 0.9% NaCl Blocking buffer: 100 mL Tris buffered saline 5 gm BSA 0.1 mL Tween 20 Peptide Competition Experiment Phosphorylation Site Specific Antibodies (PSSAs) have been developed to enable the specific and sensitive detection of phosphorylation of particular amino acid residues in target proteins, while circumventing the need for protein purification, phosphopeptide mapping or handling radioactivity. The specificity of a PSSA in each experimental system can be confirmed through peptide competition. In this technique, aliquots of antibody are pre-incubated with peptide containing the sequence of the phosphopeptide immunogen used to raise the PSSA and the corresponding non-phosphopeptide. Following preincubation with the peptide, each antibody preparation is then used as a probe in antibody-based detection methods, such as Western blotting, immunocytochemistry, flow cytometry, or ELISA. With a PSSA specific for the phosphorylated target protein, pre-incubation with an excess of peptide containing the sequence of the phosphopeptide immunogen will block all antigen binding sites, while pre-incubation with the corresponding non-phosphopeptide will not affect the antibody. In performing the Peptide Competition Experiment, it is important to note that the optimal dilutions of both antibody and peptide should be determined empirically for each specific application. The optimal dilution of antibody in these procedures is below saturating, as determined by previous experiments in your system. If an optimal antibody dilution has not been determined in your system, please refer to the Suggested Working Dilution on the antibody Product Analysis Sheet for guidance on an appropriate starting dilution. The optimal dilution of peptide used in these procedures will depend on the overall affinity or avidity of the antibody, as well as the quantity of the target antigen. A 50-150 fold molar excess of peptide to antibody is found to be effective for most peptide competition experiments. In the example presented below, the PSSA is used as a dilution of 1:1000 and the peptides are used at a concentration of 333 nM. The total volume of the phosphopeptide and non-phosphopeptide-pre-incubated antibody preparations is 2 mL, sufficient for probing Western blot strips, as well as for use in other antibody-based detection methods. Under these conditions, the molar excess of peptide to antibody is > / = 50. Procedure: 1.Prepare three identical test samples, such as identical PVDF or nitrocellulose strips to which the protein of interest has been transferred. The test samples should be blocked using a blocking buffer, such as Tris buffered saline supplemented with 0.1% Tween 20, and either 5% BSA or 5% non-fat dried milk. 2. Prepare 6.5 mL of working antibody stock solution (1:1000 in this example) by adding 6.5 µL of antibody stock solution to 6.5 mL of buffer containing blocking protein, such as TBS supplemented with 0.1% Tween 20, and either 3% BSA or 3% non-fat dried milk. 3.Apportion the unused PSSA into working aliquots and store at -20°C for future use (the stock PSSA contains 50% glycerol and will not freeze at this temperature). 4. Allow lyophilized control peptides to reach room temperature, ideally under desiccation. 5. Reconstitute each of the control peptides to a concentration of 66.7 µM with nanopure water. For a peptide with a molecular mass of 1500 , reconstitution with 1 mL water yields a solution with a concentration of 66.7 µM. 6. Apportion the unused reconstituted peptide solutions into working aliquots and store at -20°C for future use. 7. Label 3 test tubes as follows: tube 1: water only no peptide control tube 2: phosphopeptide tube 3: non-phosphopeptide 8.Into each tube, pipette the following components tube 1: 2 mL diluted PSSA solution plus 10 µL nanopure water tube 2: 2 mL diluted PSSA solution plus 10 µL phosphopeptide tube 3: 2 mL diluted PSSA solution plus 10 µL non-phosphopeptide 9.Incubate the three tubes for 30 minutes at room temperature with gentle rocking. During this incubation, the peptides have the chance to bind to the combining site of the antibody. 10.At the end of the incubation step, transfer the contents of each of the three tubes to clean reaction vessels containing one of the three identical test samples. For Western blotting strips: Incubate the strips with the pre-incubated antibody preparations for 1 hour at room temperature or overnight at 4°C. Wash each strip four times, five minutes each, to remove unbound antibody. Transfer each strip to a new solution containing a labeled secondary antibody [e.g., goat F(ab')2 anti-rabbit IgG alkaline phosphatase conjugate or goat F(ab')2 anti-rabbit IgG horseradish peroxidase conjugate. Remove unbound secondary antibody by thorough washing, and develop the signal using your chemiluminescent reagents and instrumentation. The signal obtained with antibody incubated with the Water Only, No Peptide Control (Tube 1), represents the maximum signal in the assay. This signal should be eliminated by pre-incubation with the Phosphopeptide (Tube 2), while pre-incubation with the Non-Phosphopeptide (Tube 3) should not impact the signal. If the Phosphopeptide only partially eliminates the signal, repeat the procedure using twice the volume of water or peptide solutions listed in Step 8. If partial competition is seen following pre-incubation with the Non-Phosphopeptide, repeat the procedure using half the volumes of water or peptide solutions listed in Step 8. |
| Pictures | Peptide Competition
Lysates from PC12 cells unstimulated (1) or stimulated with 0.5 M sorbitol for 5 minutes (2-6) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer overnight at 4oC, and then incubated with the ERK1&2 [pTpY185/187] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following its prior incubation with: no peptide (1, 2), the non-phosphopeptide corresponding to the phosphopeptide immunogen (3), a generic phosphothreonine-containing peptide (4), a generic phosphotyrosine-containing peptide (5), or the phosphopeptide immunogen (6). After washing, the membrane was incubated with goat F(ab)2 anti-rabbit IgG HRP conjugate and signals were detected using the Pierce SuperSignal?? method. The data show that only the phosphopeptide corresponding to ERK1&2 [pTpY185/187] blocks the antibody signal, demonstrating the specificity of the antibody. The data also show the induction of ERK1&2 [pTpY185/187] phosphorylation by the addition of sorbitol in this cell system. |
12 Secondary Antibodies
| Catalog No. | Name / Host | Presentation | Reactivity | ||||
|---|---|---|---|---|---|---|---|
| R1364B | Rabbit IgG (H&L) | ||||||
| Goat | Biotin | 2 mg / 170,00 € | |||||
| R1364F | Rabbit IgG (H&L) | ||||||
| Goat | FITC | 2 mg / 160,00 € | |||||
| R1364T | Rabbit IgG (H&L) | ||||||
| Goat | TRITC | 2 mg / 160,00 € | |||||
| R1364TR | Rabbit IgG (H&L) | ||||||
| Goat | Texas Red | 2 mg / 170,00 € | |||||
| R1364HRP | Rabbit IgG (H&L) | ||||||
| Goat | HRP | 2 mg / 170,00 € | |||||
| R1364AP | Rabbit IgG (H&L) | ||||||
| Goat | AP | 1 mg / 200,00 € | |||||
| R1458C2 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy2 | 1 mg / 320,00 € | |||||
| R1458C3 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy3 | 1 mg / 320,00 € | |||||
| R1458C35 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy3.5 | 1 mg / 320,00 € | |||||
| R1458C5 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy5 | 1 mg / 320,00 € | |||||
| R1458C55 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy5.5 | 1 mg / 320,00 € | |||||
| R1427R | Rabbit IgG (H&L) F(ab')2 Fragment multi-species ads. | ||||||
| Donkey | PE | 1 ml / 330,00 € | |||||
