![<b>Peptide Competition</b> Lysates from Jurkat cells either untreated (1, 6), treated with hydrogen peroxide (2-5), or the ionophore A23187 (7) were resolved on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 3% milk-TBST buffer for one hour at room temperature, then were incubated with the ETS1[pS251] antibody overnight at 4°C in a 1% milk-TBST buffer, following prior incubation with: no peptide (1, 2, 6, 7), the non-phosphopeptide corresponding to the immunogen (3), a generic phosphoserine-containing peptide (4), or the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F(ab')2 anti-rabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignalTM method. The data show that only the peptide corresponding to ETS1 [pS251] blocks the signal, verifying the specificity of the antibody. In addition, antibody blocking was not seen using phosphopeptides to the pS257, pS282, pS285, or pSpS309/312 sites (not shown).](/media/images/0094634.jpeg)
BP7061 ETS1 (pSer251) antibody
see related secondary antibodiessee all 22 ETS1 products
0.1 ml / 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 ETS1 (pSer251)
Synonyms
ETS-1, p54, EWSR2, , Protein C-ets-1, v-ets Erythroblastosis Virus E26 Oncogene Homolog 1 (avian)
Product review
Please read our product review about ETS1 (pSer251): Antibodies to ETS1 and ETS2 oncoproteins.
Product Description
Rabbit anti ETS1 (pSer251) , Presentation: Aff - Purified. Product is tested for Western blot / Immunoblot ( WB )
Properties
| Applications | Western blot / Immunoblot ( WB ) |
| Reactivity | Human ( Hu ), Mouse ( Ms ), Rat ( Rt ), Rabbit ( Rb ), Chicken ( Chk ), Xenopus ( Xen ), Fish ( Fish ) |
| Presentation | Aff - Purified |
| Host | Rabbit |
| Catalog Number | BP7061 |
| Swiss Prot. No. | P14921 |
| Quantity | 0.1 ml |
| Price | US$ 515 |
| Delivery | Worldwide |
| Manufacturer | Acris Antibodies GmbH |
| Datasheet | view  BP7061.pdf |
Datasheet Extract
| Background | The proto-oncogene ETS1 (E-twenty-six-specific sequence) is the founding member of a family of transcription factors that share a highly conserved DNA-binding domain, called the ETS domain, which recognizes a nucleotide motif with a GGAA/T core sequence. The ETS genes are dispersed on two separate chromosomal loci called ets-1, which codes for a
54 kDa protein, and ets-2, which codes for a 56 kDa protein. They are responsible for the regulation of critical genes involved in cell proliferation, differentiation, lymphoid development, motility, invasion, angiogenesis, and apoptosis. ETS1 has been shown to be phosphorylated on serine 251, 257, 282, and 285 by calmodulin-dependent kinase II in vitro and following antigenic stimulation of T or B lymphocytes or treatment with calcium ionophores in vivo. Phosphorylation at these sites, which are adjacent to the DNA binding domain, has been shown to inhibit ETS1 binding to specific DNA sequences but does not affect ETS1 localization to the nucleus. |
| Immunogen | Chemically synthesized phosphopeptide derived from the region of human ETS1 that contains serine 251. Swiss Num.: P14921 |
| Format | State: Liquid Ig fraction Purification: Immunoaffinity chromatography. The antibody has been negatively pre-adsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated ETS. The final product is generated by affinity chromatography using an ETS1-derived peptide that is phosphorylated at serine 251. 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, containing 0.05 % sodium azide as preservative |
| Applications | Western blot (1:1000).
Positive control: Jurkat cells treated with hydrogen peroxide or A23187. |
| Specificity | This antibody detects ETS1. Species: Human, chicken, pufferfish (Tetraodon fluviatilis), frog (Xenopus laevis), rat, mouse, rabbit. |
| Storage | Store at 2 - 8 °C up to one week or (in aliquots) at -20 °C for longer. Centrifuge vial before opening. Avoid repeated freezing and thawing.
Shelf life: one year from despatch. |
| References | Liu, H., et al. (2004) AML1/Runx1 recruits calcineurin to regulate granulocyte macrophage colony-stimulating factor by Ets1 activation. J. Biol. Chem. 279:29398-29408.
Liu, H. and Grundstrum, T. (2002) Calcium regulation of GM-CSF by calmodulin-dependent kinase II phosphorylation of Ets1. Mol. Biol. Cell. 13:4497-4507. Yordy, J.S. and R.C. Musise-Helmericks (2000) Signal transduction and the Ets family of transcription factors. Oncogene 19:6503-6513. Cowley, D.O. and B.J. Graves (2000) Phosphorylation represses Ets-1 DNA binding by reinforcing autoinhibition. Genes Dev. 14:366-376. Rabault, B. and J. Ghysdael (1994) Calcium-induced phosphorylation of ETS1 inhibits its specific DNA binding activity. J. Biol. Chem. 269:28143-28151. Fleischman, L.F., et al. (1993) c-Ets-1 protein is hyperphosphorylated during mitosis. Oncogene 8:771-780. Fisher, C.L., et al. (1991) Ligation of membrane Ig leads to calcium-mediated phosphorylation of the proto-oncogene product, Ets-1. J. Immunol. 146:1743-1749. Pognonec, P., et al., (1988) Mitogenic stimulation of thymocytes results in the calcium-dependent phosphorylation of c-ets-1 proteins. EMBO J. 7:977-983. |
| 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. 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 ultracentrifugedat 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 1% milk and 0.1% Tween 20 overnight at 4°C. 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. Cell Lysis Buffer Formulation: 10 mM Tris, pH 7.4 100 mM NaCl 1 mM EDTA 1 mM EGTA 1 mM NaF 20 mM Na4P2O7 2 mM Na3VO4 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 Formulation: 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 Formulation: 20 mM Tris-HCl, pH 7.4 0.9% NaCl Blocking Buffer Formulation: 100 mL Tris buffered saline 5 gm BSA 0.1 mL Tween 20 |
| Pictures | Peptide Competition Lysates from Jurkat cells either untreated (1, 6), treated with hydrogen peroxide (2-5), or the ionophore A23187 (7) were resolved on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 3% milk-TBST buffer for one hour at room temperature, then were incubated with the ETS1[pS251] antibody overnight at 4°C in a 1% milk-TBST buffer, following prior incubation with: no peptide (1, 2, 6, 7), the non-phosphopeptide corresponding to the immunogen (3), a generic phosphoserine-containing peptide (4), or the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F(ab')2 anti-rabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignalTM method. The data show that only the peptide corresponding to ETS1 [pS251] blocks the signal, verifying the specificity of the antibody. In addition, antibody blocking was not seen using phosphopeptides to the pS257, pS282, pS285, or pSpS309/312 sites (not shown). |
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 | |||||
Click here to see all secondary antibodies for 'BP7061 ETS1 (pSer251)'.
